Novel human K+ ion channel and therapeutic applications thereof

ABSTRACT

The present invention relates to a novel human K+ion channel, to nucleic acid molecules encoding the same and to vectors comprising said nucleic acid molecules. The invention additionally relates to antibodies specifically directed to the novel K+ion channel and to pharmaceutical compositions and diagnostic kits containing at least one of the above-mentioned components. Furthermore, the present invention relates to methods of treating a disease caused by malfunction of the polypeptide of the present invention or by the (over)expression of the nucleic acid molecule of the invention comprising administering an inhibitor of said (over)expression or of ion channel function or an inhibitor abolishing said malfunction to a patient in need thereof. Methods of devising drugs for treating or preventing the above-mentioned disease, methods of inhibiting cell proliferation and methods of prognosing cancer are additional embodiments comprised by the present invention. The invention also envisages specific antisense or gene therapies on the basis of the nucleic acid molecule of the invention for inhibiting undesired cellular proliferation, for example, in connection with cancer or in neurodegenerative diseases.

[0001] The present invention relates to a novel human K⁺ ion channel, tonucleic acid molecules encoding the same and to vectors comprising saidnucleic acid molecules. The invention additionally relates to antibodiesspecifically directed to the novel K⁺ ion channel and to pharmaceuticalcompositions and diagnostic kits containing at least one of theabove-mentioned components. Furthermore, the present invention relatesto methods of treating a disease caused by malfunction of thepolypeptide of the present invention or by the (over)expression of thenucleic acid molecule of the invention comprising administering aninhibitor of said (over)expression or of ion channel function or aninhibitor abolishing said malfunction to a patient in need thereof.Methods of devising drugs for treating or preventing the above-mentioneddisease, methods of inhibiting cell proliferation and methods ofprognosing cancer are additional embodiments comprised by the presentinvention. The invention also envisages specific antisense or genetherapies on the basis of the nucleic acid molecule of the invention forinhibiting undesired cellular proliferation, for example, in connectionwith cancer or in neurodegenerative diseases.

[0002] Potassium channels are a relevant factor in the regulation of theresting potential of cells, and this has been regarded as their majorrole in excitable and non-excitable tissues. On the other hand, theexplanation for their ubiquitous presence and the impressive variabilityin their properties remains elusive. A reasonable hypothesis is thatpotassium channels are present in all cell types because they have inaddition some “housekeeping” role, for example in cell proliferation¹.Their implication in the regulation of the cell division cycle has beentested repeatedly, and some experimental evidence has beenpresented^(2,3). However, especially since both depolarization andhyperpolarization of the membrane potential during cell cycle have beenreported as depending on cell type^(1,4), there is no general model toexplain the function of potassium channels in cell cycle. Two mechanismshave been proposed to explain the role of K⁺ channels: they eitherinfluence the intracellular Ca²⁺ concentration, or control cell volume(17, 18). Both mechanisms would indirectly influence cell proliferation.A member of the eag family has also been proposed to be preferentiallyexpressed in cancer cells (19) Several potassium channel blockers havebeen tested for their capability to block cancer cell proliferation, andsome of them have even been used as coadjuvants for tumor chemotherapy,specially in multidrug-resistant tumors. Nevertheless, the lack ofidentification of a particular potassium channel directly involved inthe control of cell proliferation has, up to date, precluded thedescription of more specific and effective treatment protocols.

[0003] Thus, the technical problem underlying the present invention wasto identify a biological component within the conglomerate of potassiumchannels with their various effects on cell cycle division that allowsan unambiguous assignment to cellular proliferation, with a specificview to human cellular proliferation. The solution to said technicalproblem is achieved by providing the embodiments characterized in theclaims.

[0004] Accordingly, the present invention relates to a nucleic acidmolecule comprising a nucleic acid molecule encoding a (poly)peptidehaving a function of the human K⁺ ion eag channel which is

[0005] (a) a nucleic acid molecule comprising a nucleic acid moleculeencoding the polypeptide having the amino acid sequence of SEQ ID: No 3or 4;

[0006] (b) a nucleic acid molecule comprising the nucleic acid moleculehaving the DNA sequence of SEQ ID: No 13 or 14;

[0007] (c) a nucleic acid molecule hybridizing to the complementarystrand of a nucleic acid molecule of (a) or (b); or

[0008] (d) a nucleic acid molecule being degenerate to the sequence ofthe nucleic acid molecule of (c).

[0009] The nucleic acid molecule of the invention encodes a(poly)peptide which is or comprises the human homologues of the rat eagchannel. In this regard the term “a nucleic acid molecule comprising anucleic acid molecule encoding a (poly)peptide having a function of thehuman K⁺ ion eag channel” may mean that said first mentioned nucleicacid molecule solely encodes said (poly)peptide. Thus, it may beidentical to said second mentioned nucleic acid molecule. Alternatively,it may comprise regulatory regions or other untranslated regions. In afurther embodiment, said first mentioned nucleic acid may compriseheterologous nucleic acid which may encode heterologous proteinaceousmaterial thus giving rise, e.g., to fusion proteins. It is further to benoted that the DNA sequences of SEQ ID NO: 13 and 14 are splice variantsof the nucleic acid sequence encoding the (poly)peptide of theinvention. The corresponding amino acid sequences are depicted in SEQ IDNO: 3 and 4.

[0010] The term “having a function of a human K⁺ ion eag channel”, asused in connection with the present invention, has the followingmeaning: The channel has a single channel conductance in asymmetricalpotassium, at 0 vM of about 6 pS. This value clearly distinguishes thehuman channel from the rat channel for which a value of about 7 pS wasmeasured. In addition or in the alternative, the above term may have thefollowing meaning: The channel has a IC50 of about 1 mM to quinidinewhen expressed in Xenopus laevis oocytes, as compared to 400μM for reag.Further, when measuring voltage-dependence of activation in highextracellular potassium using a two-electrode voltage-clamp it was foundthat in a conductance-voltage plot, the voltage for half-activation isshifted by about 40 vM or more to the right in the heag channel withrespect to the reag channel (see FIG. 13). On the basis of the abovefeatures, either alone or in combination, a differentiation based onfunction between the human ion channel of the invention and the priorart channels, in particular of the rat ion channel, is possible for theperson skilled in the art without further ado. Preferably, the channelhas all recited functions. The above values refer to values that areobtainable with the experimental set-up described in this specification.Alterations of experimental parameters such as the employment of adifferent expression system may, as is well known to the person skilledin the art, also change the above values. Yet, these embodiments arealso comprized by the scope of the present invention.

[0011] The term “hybridizing” as used in accordance with the presentinvention relates to stringent or non-stringent hybridizationconditions. Preferably, it relates to stringent conditions. Saidhybridization conditions may be established according to conventionalprotocols described, for example, in Sambrook, “Molecular Cloning, ALaboratory Manual”, Cold Spring Harbor Laboratory (1989) N.Y., Ausubel,“Current Protocols in Molecular Biology”, Green Publishing Associatesand Wiley Interscience, N.Y. (1989), or Higgins and Hames (eds) “Nucleicacid hybridization, a practical approach” IRL Press Oxford, WashingtonD.C. (1985). Hybridizing molecules or molecules falling underalternative (d), supra, also comprise fragments of the moleculesidentified in (a) or (b) wherein the nucleotide sequence need not beidentical to its counterpart in SEQ ID 13 or 14, said fragments having afunction as indicated above.

[0012] An example of one such stringent hybridization condition ishybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at65° C. for one hour. Alternatively, an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC at 42° C. Examples of suchnon-stringent hybridization conditions are 4×SSC at 50° C. orhybridization with 30-40% formamide at 42° C. Complementary strands ofhybridizing molecules comprise those which encode fragments, analoguesor derivatives of the polypeptide of the invention and differ, forexample, by way of amino acid and/or nucleotide deletion(s),insertion(s), substitution(s), addition(s) and/or recombination(s) orany other modification(s) known in the art either alone or incombination from the above-described amino acid sequences or theirunderlying nucleotide sequence(s). Using the PESIND program (Rogers,Science 234 (1986), 364-368), PEST sequences (rich in proline, glutamicacid, serine, and threonine) can be identified, which arecharacteristically present in unstable proteins. Such sequences may beremoved from the polypeptide of the invention in order to increase thestability and optionally the activity of the proteins. Methods forintroducing such modifications in the nucleic acid molecules accordingto the invention are well-known to the person skilled in the art. Theinvention also relates to nucleic acid molecules the sequence of whichdiffers from the nucleotide sequence of any of the above-describednucleic acid molecules due to the degeneracy of the genetic code. Allsuch fragments, analogues and derivatives encoding the protein of theinvention are included within the scope of the present invention, aslong as the essential characteristic immunological and/or biologicalproperties as defined above remain unaffected in kind, that is the novelnucleic acid molecules of the invention include all nucleotide sequencesencoding proteins or peptides which have at least a part of the primarystructural conformation for one or more epitopes capable of reactingwith antibodies to said polypeptide which are encoded by a nucleic acidmolecule as set forth above and which have comparable or identicalcharacteristics in terms of biological activity. Part of the inventionis therefore also concerned with nucleic acid molecules encoding apolypeptide comprising at least a functional part of the aboveidentified polypeptide encoded by a nucleic acid sequence comprised in anucleic acid molecule according to the invention.

[0013] The present inventors have recently described a potassium channel(reag) which is strongly downregulated immediately after the activationof cyclin dependent kinases (key molecules in the cell cycleregulation), in both G1-S and G2-M transitions⁵. The K⁺ current isinhibited following activation of cyclin-dependent kinases due to avoltage-dependent sodium block, which is not apparent in all phases ofthe cell cycle. The experiments presented here are aimed to determinewhether eag, in addition to being regulated by the cell cycle, is alsoable to directly influence cell proliferation and growth (20). Inaccordance with the present invention and with a view to the developmentof a suitable system for assessing (disease-related) proliferation inhuman cells, it was further attempted to study whether the implicationof the channel in the cell cycle goes in both directions, such that itis not only regulated by but also regulator of the progression of thecell cycle.

[0014] The results obtained in this rat derived ion channel system showthat in three different cell lines obtained from different species(Chinese hamster -CHO-, human-HEK293-, and mouse -NIH3T3-), the rate ofproliferation is faster when the channel is overexpressed aftertransfection of the cells with a plasmid containing the channel DNAunder the control of the cytomegalovirus promoter. FIG. 1 and FIG. 18ashow the increase in metabolic activity in cultures of CHO cells in thepresence of normal concentrations of fetal calf serum (10% FCS). Underthese normal conditions, reag transfected cells grow several foldsfaster than untransfected cells (WT).

[0015]FIG. 2 shows a comparable experiment at very low concentrations offetal calf serum (0.5% FCS). These low serum concentrations do not allowwild-type cells to grow; after a few hours, the cells start to die.However, reag transfected cells are able to proliferate under the sameconditions. The ability to overcome the growth arrest induced by theabsence of growth factors is one of the typical properties of malignanttransformation (cf FIG. 18).

[0016] Not only the metabolic activity can be used to trace theproliferation in culture. The measurement of DNA synthesis is a moredirect estimation of the rate of cell growth, since only cells enteringS phase (committed to divide) synthesize DNA. Also DNA synthesis becomesserum-independent in reag transfected cells, i.e., the growth ismaintained in the absence of growth factors (while it induces theprogrammed death of non-transfected cells). This is depicted in FIG. 3,were the incorporation of 5-Bromo-2′-deoxyuridine⁷⁻¹⁰ (BrdU) was used tomonitor DNA synthesis in the presence of 10 or 0.5% FCS in CHO cells. Asopposed to wild-type or cells transfected with an inactivatingvoltage-dependent potassium channel from rat brain (Kv1.4), there are nosignificant differences in the amount of DNA synthesized in the presenceof normal or low FCS concentrations in reag -expressing cells. Similarexperiments were done using epidermal growth factor (EGF) in HEK-293cells or platelet-derived growth factor (PDGF) in CHO cells, withessentially the same result. The pure growth factors were used to avoidthe complexity introduced by the use of whole serum.

[0017] To test the effects of eag on cell proliferation more directly,DNA synthesis was measured through incorporation of 5-Bromo-deoxyuridine(BrdU) in cells synchronized in the S-phase of the cell cycle by meansof thymidine arrest (23). Consistent with the above mentioned findings,when the S-phase of the cell cycle was allowed to proceed, reagexpressing CHO cells (CHOrEAG) showed higher metabolic activity (FIG.18B) and increased BrdU incorporation (FIG. 18C). These results suggestthat more eag-transfected cells entered the S-phase during the arrestedperiod and/or DNA synthesis was elevated, in any case indicating afaster proliferation rate in CHOrEAG cells. In the presence of lowserum, BrdU incorporation was significantly higher in CHOrEAG than inwild type cells (FIG. 18C).

[0018] Yet another cell line, NIH3T3, has been frequently used for tumortransformation assays, since these cells are very stronglycontact-inhibited, (i.e., their growth is stopped when the culturereaches confluency). This results in a homogeneous monolayer inwild-type cells. The malignant transformation of the line (throughoncogene expression) usually induces the loss of this property, andNIH3T3 cells start forming colonies composed of several layers of cells.This can be seen after the transfection with reag DNA, which induced theformation of such foci in several independent clones (FIG. 4A and B).Another standard test for transforming activity is the ability of NIH3T3cells to grow in colonies when no substrate for attachment is available.To test this, cells are plated in an agar-containing medium, where theagar will prevent contacts between the cells and the surface of theplate. Under these conditions, wild-type NIH3T3 cells were unable togrow, while cells expressing reag formed large colonies even detectableby simple visual inspection of the plate. Table I shows that reag- (butnot rKv1.4-) transfected cells formed colonies in a semisolid mediumcontaining 0.3% agar (24,25), regardless of the vector used fortransfection (FIG. 14). All of the above results indicate a transformingpotential of eag.

[0019] Altogether, the results obtained from transfected cells indicatethat reag can, at least under certain conditions, display oncogenicproperties.

[0020] Once the transforming ability of reag was determined inaccordance with the invention, the expression of the respective channelin human cancer cells was investigated. For this investigation, the cellline MCF-7 was used, which was initially obtained from a pleuraleffusion of a breast adenocarcinoma. The line is estrogen receptorpositive as well as estrogen-sensitive and relatively welldifferentiated. The strategy followed was first to testelectrophysiologically and pharmacologically for the presence of afunctional current similar to eag, and then to try an identification ofthe corresponding channel at the molecular level. However, conventionalapproaches for such an identification failed.

[0021] Namely, in most cells, the current density was too low to allowreliable measurements of the whole cell current. Low current densityprecluded an accurate measurement of channel properties using a wholecell configuration for the patch champ. Therefore, due to said lowcurrent densities encountered, another approach was resorted to. Due tosuch a low number of channels per cell, it is only possible tocharacterize the functional properties of a channel by a special patchchamp method, excising patches of membranes containing one (or a few)channels and allowing characterization on a single molecule level. Thisapproach relied on single-channel measurements in order to also compareproperties at the single-molecule level such as single channelconductance, pharmacological properties, voltage dependence, and meanopen times. Indeed, a channel with several properties compatible withreag in terms of kinetics, voltage-dependence, and pharmacology in mostmembrane patches could thus be identified. FIG. 5 shows whole-cellcurrents obtained from a MCF7 cell under nystatin patch conditions andsingle channel currents, together with their current-voltagerelationship. Despite differences in kinetics at very depolarizedvoltages, the voltage dependence of the channel in human cells is highlyreminiscent to the voltage-dependence of the reag channel. Moreover, thesingle channel properties of the putative human-eag are also verysimilar to those of reag.

[0022] Furthermore, standard approaches to isolate the said channel on amolecular level also were not successful. Several other groups haveattempted and/or are still attempting to isolate the gene coding for ahuman eag without success and this in spite of the fact that the rat eagchannel has already been published in 1994. For example, Warmke andGanetzky (Proc. Natl. Acad. Sci. USA 91 (1994), 3428-3442) specificallyset out to clone the human eag gene using conventional technology. Theywere, however, unsuccessful and cloned a novel, eag related gene whichthey termed h-erg (also referred to as HERG). Further, Wymore et al.,Circulation Res. 80 (1997), 261-268, reported that no eag specificclones could be detected in a cDNA library from human heart in spite ofthe fact that primers for amplification were used that were conservedacross the entire eag/erg superfamily. Thus, the standard approach withdegenerated oligonucleotides based on the sequence of members of thefamily revealed itself unsuccessful, although HERG was systematicallydetected by other researchers in the field. Significantly, most of theseapproaches to clone the human eag gene were made with brain libraries.The conclusion from these combined prior art data was that the human eaggene could not be cloned by conventional technology using the mostobvious source, namely brain tissue. The repeated isolation of HERGclones instead is most probably due to the relative abundance of HERGtranscripts in brain libraries, and also to the high homology betweenthe two channels. Consequently, a different strategy had to be devisedto direct the screening more specifically to eag channels. First, asdescribed herein above, a cell line expressing a channel functionallysimilar to reag was identified. Then degenerated oligonucleotides basedon conserved sequences between rat, bovine and mouse eag, but divergentfrom HERG were designed. Using these primers, the cDNA obtained fromMCF7 cells by PCR was amplified, and a band of the expected size wascloned in a suitable vector and sequenced. The amplified fragmentcorresponded to approximately 400 bp within the core region of thechannel protein, and shared 90% identity to the reag sequence at the DNAlevel, and 99% at the amino acid level. However, at this stage it wasstill quite unclear what the thus identified clone corresponded to. Forexample, it was quite possible that a further member of the eag familyhad been identified. This is in particular true in view of the fact thatdespite of a number of attempts with brain libraries, nobody had beenable to clone the human eag gene and that the MCF7 line is a breastcancer derived line.

[0023] Since MCF7 cells are immortal cells, it is assumed that a numberof genes is mutated. Ab initio, it could have been expected that thehuman eag channel, if at all expressed in this cell line, was mutated.Under this assumption, it was quite uncertain whether this cell linecould at all be used for the isolation of the desired gene.

[0024] Due to the prior art failures to clone human eag gene from brainlibraries and the above recited uncertainties with immortalized celllines, another source for a library was in need. The 400 bp fragment wastherefore used to screen a normal human breast cDNA library. Due to thepresence of eag in breast cancer cells, such a library was expected tocomprise heag clones. Surprisingly, however, after screening 2×10⁶phages, no human-eag clones could be identified in said library. Thisrises the possibility that the channel is expressed only in tumor cells,and not in normal tissue. Specific oligonucleotides, namely5′-CCAAACACACACACCAGC, 5′-CGTGGATGTTATCTTTTTGG to check for heagfragments by PCR amplification directly from the above library weredesigned, but no evidence for the presence of any eag clones in thislibrary was found. In view of the above discussed prior art results, itcame as a further surprise that the same primers detected heag in anormal human brain cDNA library, that was therefore screened. First, theprobe obtained from MCF7 cells was used to check 10⁶ phages. Thisprocedure allowed to isolate a 1.6 kbp fragment from human eag. Thisfragment was then used as a probe for the screening of 2×10⁶ phages fromthe same library. Several independent clones were isolated, but none ofthem was a full-length clone. Furthermore, only one clone contained the5′ end of the sequence, while two of them contained the 3′ end and partof the 3′ non-coding region. It is likely that the abundance ofrestriction sites in the nucleic acid sequence encoding the channel hasinduced this extensive fragmentation of the cDNA. For example, whenEcoRI was used to extract the inserts of the library that was cloned inλ-gt10 phage at the EcoRI site, this conventional approachsystematically failed to find the 5′ end of the molecule (there is anEcoRI site at position 400 of the clone). The pooled positive cloneswere therefore screened again by PCR, trying to amplify the startcondon, and only by this means it was possible to isolate one phage thatcontained this ATG. Two splice variants of heag were cloned, bothexpressed in brain tissue. The sequence obtained for heag 1 and heag 2and their deduced amino acid sequence are shown in FIGS. 10 and 11, andcompared to other members of the family.

[0025] The deduced amino acid sequence is identical to the sequencepublished after the priority date of the present invention by Occhidoro(27) and is 97,7% identical to reag. As mentioned, a second (81 bplonger) splice variant (heag 2) was also isolated analogous to thatreported for bovine and mouse eag channels (28), the splice insertionbeing identical in all three species. The chromosomal localization ofheag was determined by FISH detection (29) to map to chromosomelq32.1-32.3 (see also ref. 26).

[0026] To further check the possibility that heag is not expressed innormal mammary gland, as opposed to MCF-7 cancer cells, we performedsingle-tube RT-PCR experiments using total RNA from human brain, humanmammary gland, and MCF-7 cells (FIG. 12), using as primers twooligonucleotides designed to discriminate between the two splicevariants of heag. In human brain, two splice variants were detected,while only the short one was expressed in MCF-7 cells (this, togetherwith the lack of amplification in the absence of reverse transcriptase,rules out a possible contamination by genomic DNA of the RNApreparation). No heag signal was detected in normal mammary gland RNAwith this highly sensitive technique. This result was totallyunexpected, because preliminary results had suggested that expressionwas present in tumor cells from the same organ. Further, after Southernblot analysis of the RT-PCR products a faint band hybridizing with aheag probe in mammary gland was identified. Accordingly, it is quitedifficult to make a strong statement on the total absence of heagmessage in breast in view of these contradictory experimental data.

[0027] Furthermore, electrophyiological properties (21, 30) of heag weretested in Xenopus oocytes. As described above, they did not differsignificantly from those or reag with the above mentioned exceptions,e.g. a shift in activation of 40 vM to more depolarized potentials whenboth channels were measured under identical conditions. Theelectrophysiological observations of heag channels expressed in Xenopusoocytes correlate well to hose reported by Bijlenga et al. (31).

[0028] The present invention also relates to a nucleic acid moleculespecifically hybridizing to the nucleic acid molecule of the inventionwhich comprises the sequence 5′-GGGAGGATGACCATGGCT.

[0029] This embodiment of the present invention is particularly usefulfor specific antisense therapies for inhibiting cell proliferation aswill be discussed in more detail herein below (e.g. in Example 5). Inaddition, this embodiment of the nucleic acid molecule of the inventioncan, naturally, also be used as a probe for specifically detecting heagmRNA in tissues, for example, by employing the Northern Blot technology.The analysis of heag mRNA expression in various tissues by Northern blotrevealed a strong hybridization signal of approximately 9.2 kb in brainand a weak signal of similar size in placenta. Heart, lung, liver,skeletal muscle, kidney and pancreas were negative even following longexposures. In addition, total RNA from human brain, heart, trachea,adrenal gland, liver, kidney, skeletal muscle and mammary gland, andspinal cord poly(A)⁺ RNA, as well as total RNA from theadenovirus-transformed line 293 (a human non-tumoral cell line) wereassayed by single-tube RT-PCR and Southern blot. Under theseexperimental conditions, heag was detected in brain only, where bothsplice variants were identified (FIG. 15; Example 3).

[0030] The preferential expression of heag in brain was intriguing sincethe first cDNA had been isolated from an epithelial tumor cell line(MCF-7) and not from brain tissue (see above). To elucidate the presenceof heag in other tumoral cell lines, total RNA was prepared from HeLa(cervix carcinoma), SHSY-5Y (neuroblastoma), and lines from mammarygland tumors: COLO-824 (carcinoma), EFM-19 (carcinoma), and BT-474(ductal carcinoma). Total RNA from brain, MCF-7 cells, 293 cells and RNAfrom cultures of mammary gland epithelial cells (included to circumventthe mixed cell populations in whole mammary gland) served as controls.All cell lines were obtained from DSMZ (Deutsche Sammlung vonMikroorganismen und Zellkulturen) and maintained following the DSMZcatalog guidelines. Normal human mammary epithelial cells were purchasedfrom BioWhittaker. The primers were designed to amplify different bandsfor heag 1 and heag 2, thus allowing us to rule out false positives dueto genomic DNA contamination (controls in the absence of reversetranscriptase were also performed). HeLa, SHSY-5Y, EFM-19 and MCF-7 RNAexhibited an heag band, whereas COLO-824 and BT-474 signals wereindistinguishable from background (FIG. 15B). Cultured epithelial cellsand 293 cells (FIG. 15A) were negative. As discussed above, it could beshown in accordance with the present invention that reag transfectedcells can display oncogenic properties. Thus, to determine whether theexpression of heag is advantageous for tumor cells in vivo, subcutaneousimplants of CHO cells expressing the channel (CHOHEAG cells) into theflank of female scid (severe combined immunodeficiency, 32) mice wereperformed and it could be shown that expression of heag represents anadvantage for the proliferation of tumor cells in vivo, since CHOHEAGtumors grow faster and are more aggressive than CHOKv tumors. Thus, theembodiment of the nucleic acid molecule of the present invention may beemployed in the quantitative and qualitative analysis of the expressionlevel of human eag in various disease states detectable in a tissue thatmay be indicative of, for example, cancer (in particular mammacarcinoma, neuroblastoma), psoriasis, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, lateral amyotrophic sclerosisor multiple sclerosis.

[0031] In a preferred embodiment of the nucleic acid molecule of theinvention, said nucleic acid molecule is DNA, such as genomic DNA.Whereas the present invention also comprises synthetic or semi-syntheticDNA molecules or derivatives thereof, such as peptide nucleic acid, themost preferred DNA molecule of the invention is cDNA.

[0032] In a further preferred embodiment of the present invention, saidnucleic acid molecule is RNA, preferably mRNA.

[0033] Another preferred embodiment of the nucleic acid molecule of theinvention encodes a fusion protein. For example, the nucleic acidmolecule of the invention can be fused in frame to a detectable markersuch as FLAG or GFP.

[0034] The invention further relates to a vector, particularly plasmid,cosmids, viruses and bacteriophages comprising the nucleic acid moleculeof the invention. Such vectors may comprise further genes such as markergenes which allow for the selection of said vector in a suitable hostcell and under suitable conditions. Thus the polynucleotide of theinvention can be operatively linked in said vector to expression controlsequences allowing expression in prokaryotic or eukaryotic cells.Expression of said polynucleotide comprises transcription of thepolynucleotide into a translatable mRNA. Regulatory elements ensuringexpression in eukaryotic cells, preferably mammalian cells, are wellknown to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers. Possible regulatory elementspermitting expression in prokaryotic host cells comprise, e.g., the lac,trp or tac promoter in E. coli, and examples for regulatory elementspermitting expression in eukaryotic host cells are the AOX1 or GAL1promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcomavirus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian andother animal cells. Beside elements which are responsible for theinitiation of transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site, downstream of the polynucleotide. In this context,suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1 (GIBCO BRL).

[0035] Preferably, said vector is an expression vector and/or a genetransfer or targeting vector. Expression vectors and gene targeting ortransfer vectors are well-known in the art and can be adapted forspecific purposes of the invention by the person skilled in the art.Thus, expression vectors derived from viruses such as retroviruses,vaccinia virus, adeno-associated virus, herpes viruses, or bovinepapilloma virus, may be used for delivery of the polynucleotides orvectors of the invention into targeted cell populations. Methods whichare well known to those skilled in the art can be used to constructrecombinant viral vectors; see, for example, the techniques described inSambrook, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory (1989) N.Y. and Ausubel, Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y.(1989). Alternatively, the polynucleotides and vectors of the inventioncan be reconstituted into liposomes for delivery to target cells.

[0036] The invention furthermore relates to a host transformed with thevector of the invention. Said host may be a prokaryotic or eukaryoticcell; see supra. The polynucleotide or vector of the invention which ispresent in the host cell may either be integrated into the genome of thehost cell or it may be maintained extrachromosomally. In this respect,it is also to be understood that the recombinant DNA molecule of theinvention can be used for “gene targeting” and/or “gene replacement”,for restoring a mutant gene or for creating a mutant gene via homologousrecombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87(1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, OxfordUniversity Press. Preferably, the host is a mammalian cell, a fungalcell, a plant cell, an insect cell or a bacterial cell. Preferred fungalcells are, for example, those of the genus Saccharomyces, in particularthose of the species S. cerevisiae. The term “prokaryotic” is meant toinclude all bacteria which can be transformed or transfected with apolynucleotide for the expression of the protein of the presentinvention. Prokaryotic hosts may include gram negative as well as grampositive bacteria such as, for example, E. coli, S. typhimurium,Serratia marcescens and Bacillus subtilis. Methods for preparing fused,operably linked genes and expressing them in bacteria or animal cellsare well-known in the art (Maniatis, et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY, 1989). The genetic constructs and methods described therein can beutilized for expression the protein of the present -invention inprokaryotic hosts. In general, expression vectors containing promotersequences which facilitate the efficient transcription of the insertedpolynucleotide are used in connection with the host. The expressionvector typically contains an origin of replication, a promoter, and aterminator, as well as specific genes which are capable of providingphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be grown in fermentors and cultured according totechniques known in the art to achieve optimal cell growth. Thepolypeptides of the invention can then be isolated from the grownmedium, cellular lysates, or cellular membrane fractions. The isolationand purification of the microbially or otherwise expressed polypeptidesof the invention may be by any conventional means such as, for example,preparative chromatographic separations and immunological separationssuch as those involving the use of monoclonal or polyclonal antibodies.As regards mammalian cells, HEK 293, CHO, HeLa and NIH 3T3 arepreferred. As regards insect cells, it is most preferred to useSpodoptera frugiperda cells, whereas the most preferred bacterial cellsare E.coli cells.

[0037] The invention also relates to a method of producing the(poly)peptide encoded by the nucleic acid molecule of the inventioncomprising culturing the host of the invention and isolating theproduced (poly)peptide.

[0038] Depending on the vector constructing employed, the (poly)peptideof the invention may be exported to the culture medium or maintainedwithin the host. Suitable protocols for obtaining the (poly)peptideproduced are well-known in the art for both ways of (poly)peptideproduction.

[0039] The present invention furthermore relates to a (poly)peptideencoded by the nucleic acid molecule of the invention or produced by themethod of the invention. The new channel is envisaged to show astructure having a short amino-terminal region, probably intracellular,five membrane-spanning segments, a hydrophobic hairpin entering themembrane, a sixth transmembrane segment, and a long C-terminalcytoplasmic part comprising a cyclic-nucleotide binding consensussequence, a nuclear localization consensus sequence, and a hydrophobicdomain probably forming a coiled-coil structure. The polypeptide of theinvention may also be a functional fragment of the human K⁺ ion channel.By “functional fragment” polypeptides are meant that exhibit any of theactivity of heag as described above. Using recombinant DNA technology,fragments of the (poly)peptide of the invention can be produced. Thesefragments can be tested for the desired function, for example, asindicated above, using a variety of assay systems such as thosedescribed in the present invention. Preferably, said fragments comprisethe C-terminal portion of the novel ion channel.

[0040] The present invention also relates to an antibody specificallydirected to the (poly)peptide of the invention. The antibody of theinvention specifically discriminates between the human eag channel andthe prior art channels such as mouse and rat eag and preferably binds toepitopes in the C-terminal part of the ion channel. The term “antibody”,as used in accordance with the invention, also relates to antibodyfragments or derivatives such as F(ab)₂, Fab′, Fv or scFv fragments;see, for example, Harlow and Lane, “Antibodies, A Laboratory Manual”,CSH Press 1988, Cold Spring Harbor, N.Y. Preferably, the antibody of theinvention is a monoclonal antibody.

[0041] The invention also relates to a pharmaceutical compositioncomprising the nucleic acid molecule of the invention, the vector of theinvention, the polypeptide of the invention and/or the antibody of theinvention and a pharmaceutically acceptable carrier and/or diluentand/or excipient.

[0042] Examples of suitable pharmaceutical carriers and diluents as wellas of excipients are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. These pharmaceutical compositions can beadministered to the patient in need thereof at a suitable dose.Administration of the suitable compositions may be effected by differentways, erg., by oral, intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. The dosage regimenwill be determined by the attending physician and clinical factors. Asis well known in the medical arts, dosages for any one patient dependupon many factors, including the patient's size, body surface area, age,the particular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Generally, the regimen as a regular administration of thepharmaceutical composition should be in the range of 1 μg to 10 mg unitsper day. If the regimen is a continuous infusion, it should also be inthe range of 1 μg to 10 mg units per kilogram of body weight per minute,respectively. Progress can be monitored by periodic assessment. Dosageswill vary but a preferred dosage for intravenous administration of DNAis from approximately 10⁶ to 10¹² copies of the DNA molecule. Thecompositions of the invention may be administered locally orsystemically. Administration will generally be parenterally, e.g.,intravenously; DNA may also be administered directly to the target site,e.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery.

[0043] It is envisaged by the present invention that the variouspolynucleotides and vectors of the invention are administered eitheralone or in any combination using standard vectors and/or gene deliverysystems, and optionally together with a pharmaceutically acceptablecarrier or excipient. Subsequent to administration, said polynucleotidesor vectors may be stably integrated into the genome of the subject. Onthe other hand, viral vectors may be used which are specific for certaincells or tissues and persist in said cells or tissues. Suitablepharmaceutical carriers and excipients are, as has been stated above,well known in the art. The pharmaceutical compositions preparedaccording to the invention can be used for the prevention or treatmentor delaying of different kinds of diseases, which are related to theundesired (over)expression of the above identified nucleic acid moleculeof the invention. In a preferred embodiment the pharmaceuticalcomposition comprises antisense oligodesoxynucleotides, as for exampledescribed in example 5, capable of regulating, preferably decreasingheavy expression.

[0044] Furthermore, it is possible to use a pharmaceutical compositionof the invention which comprises the polynucleotide or vector of theinvention in gene therapy. Suitable gene delivery systems may includeliposomes, receptor-mediated delivery systems, naked DNA, and viralvectors such as herpes viruses, retroviruses, adenoviruses, andadeno-associated viruses, among others. Gene therapy, which is based onintroducing therapeutic genes, for example for vaccination into cells byex-vivo or in-vivo techniques is one of the most important applicationsof gene transfer. Suitable vectors, methods or gene-delivery systems forin-vitro or in-vivo gene therapy are described in the literature and areknown to the person skilled in the art; see, e.g., Giordano, NatureMedicine 2 (1996), 534-539; Schaper, Circ. Res. 79 81996), 911-919;Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996),370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91(1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251; Verma,Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30;Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996),714-716; WO 94/29469; WO 97/00957; U.S. Pat. No. 5,580,859; U.S. Pat.No. 5,589,466; U.S. Pat. No. 4,394,448 or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640, and references cited therein. Thenucleic acid molecules and vectors of the invention may be designed fordirect introduction or for introduction via liposomes, or viral vectors(e.g. adenoviral, retroviral) into the cell. Additionally, a baculoviralsystem can be used as eukaryotic expression system for the nucleic acidmolecules of the invention. Delivery of nucleic acids to a specific sitein the body for gene therapy may also be accomplished using a biolisticdelivery system, such as that described by Williams (Proc. Natl. Acad.Sci. USA 88 (1991), 2726-2729).

[0045] Standard methods for transfecting cells with recombinant DNA arewell known to those skilled in the art of molecular biology, see, e.g.,WO 94/29469. Gene therapy may be carried out by directly administeringthe recombinant DNA molecule or vector of the invention to a patient orby transfecting cells with the polynucleotide or vector of the inventionex vivo and infusing the transfected cells into the patient.Furthermore, research pertaining to gene transfer into cells of the germline is one of the fastest growing fields in reproductive biology. Genetherapy, which is based on introducing therapeutic genes into cells byex vivo or in vivo techniques is one of the most important applicationsof gene transfer. Suitable vectors and methods for in vitro or in vivogene therapy are described in the literature and are known to the personskilled in the art; see, e.g., WO94/29469, WO 97/00957 or Schaper(Current Opinion in Biotechnology 7 (1996), 635-640) and referencescited above. The polynucleotides and vectors comprised in thepharmaceutical composition of the invention may be designed for directintroduction or for introduction via liposomes, or viral vectors (e.g.adenoviral, retroviral) containing said recombinant DNA molecule intothe cell. Preferably, said cell is a germ line cell, embryonic cell,stem cell or egg cell or derived therefrom. An embryonic cell can be forexample an embryonic stem cell as described in, e.g., Nagy, Proc. Natl.Acad. Sci. USA 90 (1993) 8424-8428.

[0046] It is to be understood that the introduced polynucleotides andvectors of the invention express the (poly)peptide of the inventionafter introduction into said cell and preferably remain in this statusduring the lifetime of said cell. For example, cell lines which stablyexpress the polynucleotide under the control of appropriate regulatorysequences may be engineered according to methods well known to thoseskilled in the art. Rather than using expression vectors which containviral origins of replication, host cells can be transformed with thepolynucleotide or vector of the invention and a selectable marker,either on the same or separate vectors. Following the introduction offoreign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective medium. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows for the selection of cells having stably integratedthe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. Such engineered cell linesare particularly useful in screening methods or methods for identifyingan inhibitor of the polypeptide of the present invention as describedbelow.

[0047] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, Cell11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adeninephosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk, hgprt oraprt cells, respectively. Also, antimetabolite resistance can be used asthe basis of selection for dhfr, which confers resistance tomethotrexate (Wigler, Proc. Natl. Acad. Sci. USA 77 (1980), 3567; OHare,Proc. Natl. Acad. Sci. USA 78 (1981), 1527), gpt, which confersresistance to mycophenolic acid (Mulligan, Proc. Natl. Acad. Sci. USA 78(1981), 2072), neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, J. Mol. Biol. 150 (1981), 1), hygro, which confersresistance to hygromycin (Santerre, Gene 30 (1984), 147), Shble, whichconfers resistance to Zeocin® (Mulsant, Somat. Cell. Mol. Genet. 14(1988), 243-252 or puromycin (pat, puromycin N-acetyl transferase).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman, Proc.Natl. Acad. Sci. USA 85 (1988), 8047); and ODC (ornithine decarboxylase)which confers resistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.).Cells to be used for ex vivo gene therapy are well known to thoseskilled in the art. For example, such cells include for example cancercells present in blood or in a tissue or preferably the correspondingstem cells.

[0048] Furthermore, the invention relates to a diagnostic compositioncomprising the nucleic acid molecule of the invention, the vector of theinvention, the polypeptide of the invention and/or the antibody of theinvention.

[0049] The diagnostic composition of the invention is useful indetecting the onset or progress of diseases related to the undesiredexpression or overexpression of the nucleic acid molecule of theinvention. As has been pointed out herein above, such diseases areinterrelated or caused by an increased or ongoing cellularproliferation. Accordingly, the diagnostic composition of the inventionmay be used for assessing the onset or the disease status of cancer.Having thus an early criterium for tumor activity, suitablecounter-measures can immediately be applied. Such an immediate actionwill, of course, significantly improve the prognosis of the patient.These considerations equally apply to the diagnosis of metastases andrecurrent tumors.

[0050] On the other hand, not all types of tumors may be characterizedby an undesired expression or overexpression of the nucleic acidmolecule of the invention. Alternatively, said (over)expression mayoccur only in certain stages, such as early stages, of tumordevelopment. Therefore, the diagnostic composition of the invention mayalso or alternatively be employed as a means for the classification oftumors or of the developmental status of a tumor. Naturally, the or mostof the applications of the composition of the invention described-herefor tumors also apply to other diseases interrelated with or caused bythe undesired (over)expression of the nucleic acid molecule of theinvention.

[0051] Furthermore, a disease as recited throughout this specificationalso could be caused by a malfunction of the polypeptide of the presentinvention. Said disease could be interrelated or caused by, for example,an increased or reduced gene dosis of the polypeptide of the presentinvention, an increased or reduced activity of said polypeptide e.g. dueto a modification in the primary amino acid sequence as compared to thecorresponding wild-type polypeptide in a cell or tissue or a loss of theregulation of the activity of said polypeptide. Said disease mightfurther be caused by an incorrect expression of the polypeptide duringcell cycle progression or cell development. For example, mutated bindingsites to intracellular or extracellular compounds, e.g. ions or secondmessengers or regulatory proteins, might result in a malfunction of thepolypeptide of the present invention as it changes the bindingcharacteristics for said compounds regulating the activity of saidpolypeptide. Malfunction could also be caused by defective modificationssites, for example, phosphorylation or glycosylation sites. It alsomight be caused by incorrect splicing events and therefore by expressionof a truncated or extended polypeptides, for example, if heag 1 isexpressed instead of heagh 2 or vice versa.

[0052] Thus, in a further embodiment the diagnostic compositiondescribed above could also be used to detect a malfunction of thepolypeptide of the present invention.

[0053] In a further embodiment, the invention relates to a method forpreventing or treating a disease which is caused by the malfunction ofthe polypeptide of the invention, comprising introducing an inhibitor ofthe expression of the nucleic acid molecule of the present invention oran inhibitor or a modifying agent of the malfunction of the(poly)peptide of the present invention or a nucleic acid molecule codingheag or a polypeptide having heag activity into a mammal affected bysaid disease or being suspected of being susceptible to said disease.Methods for introduction of a nucleic acid molecule of the presentinvention encoding heag into a cell or subject, i.e. gene therapy, aredescribed within this specification as well as methods for theidentification of inhibitors of the expression of a nucleic acidmolecule of the present invention. Furthermore, inhibitors or modifyingagents of the malfunction of the polypeptide of the present inventioncan be identified according to methods for the identification ofinhibitors inhibitors of the polypeptide of the present invention knownto a person skilled in the art (see below). For example, some geneticchanges causing a malfunction of the polypeptide of the presentinvention lead to altered protein conformational states. Mutant proteinscould possess a tertiary structure that renders them far less capable offascilitating ion transport. Restoring the normal or regulatedconformation of mutated proteins is the most elegant and specific meansto correct these molecular defects. Pharmacological manipulations thusmay aim at restoration of wild-type conformation of the protein. Thus,the polynucleotides and encoded proteins of the present invention mayalso be used to design and/or identify molecules which are capable ofactivating the wild-type function of a derivative of the polypeptide ofthe present invention displaying said malfunction.

[0054] The doses and routes for the administration for the treatment ofa patient in need thereof have already been discussed herein above inconnection with the pharmaceutical composition of the invention.Diseases that may be treated using the method of the present inventioncomprise any diseases that are correlated with cellular proliferation.Preferred diseases that fall into this category are tumor diseases suchas cancer (breast cancer, neuroblastoma etc.), psoriasis, anddegenerative diseases, especially those of the nervous system such asAlzheimer's disease, multiple sclerosis, lateral amyotrophic sclerosis,and Parkinson's disease.

[0055] Preferably, said inhibitor of the expression or overexpression ofsaid nucleic acid molecule is the nucleic acid molecule of the inventionthat hybridizes to the nucleic acid molecule encoding the ion channel ofthe invention or fragment thereof. For example, this nucleic acidmolecule can be an antisense oligodesoxynucleotide (ODN). The inventorscould show that antisense ODNs treatment significantly reduces DNAsynthesis of several tumor cells, e.g. EFM cells, SHSY-5Y cells and HeLacells (Example 5). Thus, in a preferred embodiment the nucleic acidmolecule comprises antisense ODNs.

[0056] In a further preferred embodiment, said inhibitor of polypeptidefunction is the antibody of the invention or a drug. Said drug can behistamine receptor H1 inhibitor. Preferably, said drug inhibits activeheag, for example, acts as use-dependent, probably open-channel blocker,preferably said drug is astemizole or terfenadine. Further suitabledrugs can be identified or designed by the person skilled in the art onthe basis of the teachings of the present invention. Preferably, thedrug will have an affinity to heag channel in the mM range, morepreferable in the nM range or lower. Preferably, the drug has no effecton other channels, for example on cardiac channels.

[0057] In a further preferred embodiment of the invention, said methodfurther comprises prior to the introduction step,

[0058] (a) obtaining cells from the mammal infected by said disease and,after said introduction step, wherein said introduction is effected intosaid cells,

[0059] (b) reintroducing said cells into said mammal or into a mammal ofthe same species.

[0060] This embodiment of the present invention is particularly usefulfor gene therapy purposes which will reduce the treatment durationlargely and increase the effectivity and reduce (even eliminate) sideeffects. In addition, this embodiment of the method of the invention canalso be employed in the context or in combination with conventionalmedical therapy. The removal from and the reintroduction into saidmammal may be carried out according to standard procedures.

[0061] Preferably, the above referenced cell is a germ cell, anembryonic cell or an egg cell or a cell derived from any of these cells.

[0062] The invention further relates to a method of designing a drug forthe treatment of a disease which is caused by the undesired expressionor overexpression of the nucleic acid molecule of the inventioncomprising:

[0063] (a) identification of a specific and potent drug;

[0064] (b) identification of the binding site of said drug bysite-directed mutagenesis and chimeric protein studies;

[0065] (c) molecular modeling of both the binding site in the(poly)peptide and the structure of said drug; and

[0066] (d) modifications of the drug to improve its binding specificityfor the (poly)peptide.

[0067] The term “specific and potent drug” as used herein refers to adrug that potently and specifically blocks heag function.

[0068] All techniques employed in the various steps of the method of theinvention are conventional or can be derived by the person skilled inthe art from conventional techniques without further ado. Thus,biological assays based on the herein identified features of the ionchannel of the invention may be employed to assess the specificity orpotency of the drugs wherein the decrease of one or more activities ofthe ion channel may be used to monitor said specificity or potency.Steps (b) and (d) can be carried out according to conventional protocolsdescribed, for example, in K. L. Choi, C. Mossman, J. Aubé & G. Yellen.The International Quaternary Ammonium Receptor Site of Shaker PotassiumChannels. Neuron 10, 533-541 (1993), C.-C. Shieh & G. E. Kirsch:Mutational Analysis of Ion Conduction and Drug Binding Sites in theInner Mouth of Voltage-Gated K⁺-Channels. Biophys. J. 67,2316-2325(1994), or C. Miller: The Charybdotoxin Family of K-Channel-BlockingPeptide. Neuron 15, 5-10 (1995).

[0069] For example, identification of the binding site of said drug bysite-directed mutagenesis and chimerical protein studies can be achievedby modifications in the (poly)peptide primary sequence that affect thedrug affinity; this usually allows to precisely map the binding pocketfor the drug.

[0070] As regards step (c), the following protocols may be envisaged:Once the effector site for drugs has been mapped, the precise residuesinteracting with different parts of the drug can be identified bycombination of the information obtained from mutagenesis studies (step(b)) and computer simulations of the structure of the binding site(since a potassium channel has recently been crystallized in the art,this can now be done by the person skilled in the art without furtherado) provided that the precise three-dimensional structure of the drugis known (if not, it can be predicted by computational simulation). Ifsaid drug is itself a peptide, it can be also mutated to determine whichresidues interact with other in the heag molecule.

[0071] Finally, in step (d) the drug can be modified to improve itsbinding affinity or its potency and specificity. If, for instance, thereare electrostatic interactions between a particular residue of heag andsome region of the drug molecule, the overall charge in that region canbe modified to increase that particular interaction; additionally, ifthose interactions occur with a region of heag that is not conservedwith other channel proteins, it is conceivable that an improvement ofthat interaction while other binding factors are weakened will improvethe specificity of the drug.

[0072] Identification of binding sites may be assisted by computerprograms. Thus, appropriate computer programs can be used for theidentification of interactive sites of a putative inhibitor and thepolypeptide of the invention by computer assisted searches forcomplementary structural motifs (Fassina, Immunomethods 5 (1994),114-120). Further appropriate computer systems for the computer aideddesign of protein and peptides are described in the prior art, forexample, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak,Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986),5987-5991. Modifications of the drug can be produced, for example, bypeptidomimetics and other inhibitors can also be identified by thesynthesis of peptidomimetic combinatorial libraries through successivechemical modification and testing the resulting compounds. Methods forthe generation and use of peptidomimetic combinatorial libraries aredescribed in the prior art, for example in Ostresh, Methods inEnzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996),709-715. Furthermore, the three-dimensional and/or crystallographicstructure of inhibitors of the polypeptide of the invention can be usedfor the design of peptidomimetic inhibitors, e.g., in combination withthe (poly)peptide of the invention (Rose, Biochemistry 35 (1996),12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

[0073] An exemplary strategy for identifying a specific inhibitor thatmay be used in accordance with the present invention is provided in theappended examples.

[0074] The invention also relates to a method of identifying aninhibitor of the expression of the nucleic acid of the invention or of afunction of the (poly)peptide of the invention comprising:

[0075] (a) testing a compound for the inhibition or reduction oftranslation wherein said compound is selected from antisenseoligonucleotides and ribozymes; or

[0076] (b) testing a compound for the inhibition of transcriptionwherein said compound binds to the promoter region of the gene encodingthe (poly)peptide of the invention and preferably with transcriptionfactor responsive elements thereof; or

[0077] (c) testing peptides or antibodies suspected to block theproliferative activity of the (poly)peptide of the invention for saidblocking activity.

[0078] As regards alternative (b) referred to above, it may beadvantageous to first characterize the promoter region and locatetranscription factor responsive sequences in it. Then it would bepossible to genetically manipulate the promoter to render it moresensitive to repressors or less sensitive to enhancers. Turning now toalternative (c), it may be advantageous to first locate the part orparts of the ion channel of the invention implicated in the generationof proliferation disorders. Compounds that have been positive in one ofthe test systems are, prima facie, useful as inhibitors.

[0079] Peptidomimetics, phage display and combinatorial librarytechniques are well-known in the art and can be applied by the personskilled in the art without further ado to the improvement of the drug orinhibitor that is identified by the basic method referred to hereinabove.

[0080] In a further embodiment, the present invention relates to amethod of inhibiting cell proliferation comprising applying an inhibitorto expression of the nucleic acid of the invention or the (poly)peptideof the invention. The method of the invention may be carried out invitro, ex vivo or when application is to a subject, in vivo.

[0081] The present invention also relates to a method of prognosingcancer and/or neurodegenerative diseases and/or psoriasis comprisingassessing the expression of the nucleic acid molecule of theinvention-or assessing the quantitative presence of the (poly)peptide ofthe invention. In a preferred embodiment said cancer is a mammacarcinoma or neuroblastoma, in a more preferred embodiment said canceris breast adenocarcinoma, breast carcinoma ductal type, or cervixcarcinoma. In a further embodiment said neurodegenerative diseases isAlzheimer's disease, Parkinson's disease, lateral amytrophic sclerosisor multiple sclerosis.

[0082] The method of the invention may be carried out in vitro, in vivo,or ex vivo. Suitable protocols for carrying out the method of theinvention are well-known in the art and include, as regards in vitrotechniques, Northern blotting for the assessment of the level of mRNA orthe analysis of tissue by microscopic techniques using, for example,antibodies that specifically recognize the (poly)peptide of theinvention. One or more these techniques may be combined with PCR basedtechniques which may also or in combination with further (conventional)techniques be used for the above recited assessment.

[0083] In a preferred embodiment of the above-mentioned methods of theinvention, said mammal is a human, rat or mouse.

[0084] The present invention further relates to the use of the nucleicacid molecules of the invention in gene therapy. As has been pointed outhere above, gene therapy may be designed to inhibit cell proliferationand thus treat any disease affected thereby such as cancer or psoriasisin a specific way. The invention particularly envisages two independentlines carrying out such gene therapy protocols:

[0085] (a) Mutagenesis of the channel together with chemical engineeringof Hi antagonists (preferably of astemizole) in order to obtain a drugspecific for human eag;

[0086] (b) Quantitative and qualitative analysis of the expressionlevels of eag in cancer tissue, in order to design a diagnostic and/orprognostic method. This would also allow the design of genetic therapiesagainst specific tumors.

[0087] For example, the nucleic acid molecule may be introduced in vivointo cells using a retroviral vector (Naldini et al., Science 272(1996), 263-267; Mulligan, Science 260 (1993), 926-932) or anotherappropriate vector. Likewise, in accordance with the present inventioncells from a patient can be isolated, modified in vitro using standardtissue culture techniques and reintroduced into the patient. Suchmethods comprise gene therapy or gene transfer methods which have beenreferred to herein above.

[0088] Finally, the present invention relates to a kit comprising thenucleic acid molecule specifically hybridizing to the nucleic acidmolecule encoding the (poly)peptide of the invention, the vector of theinvention, the polypeptide of the invention-and/or the antibody of theinvention.

[0089] The kit of the invention can, inter alia, be employed in a numberof diagnostic methods referred to above. The kit of the invention maycontain further ingredients such as selection markers and components forselective media suitable for the generation of transformed host cellsand transgenic plant cells, plant tissue or plants. Furthermore, the kitmay include buffers and substrates for reporter genes that may bepresent in the recombinant gene or vector of the invention. The kit ofthe invention may advantageously be used for carrying out the method ofthe invention and could be, inter alia, employed in a variety ofapplications referred to herein, e.g., in the diagnostic field or asresearch tool. The parts of the kit of the invention can be packagedindividually in vials or in combination in containers or multicontainerunits. Manufacture of the kit follows preferably standard procedureswhich are known to the person skilled in the art.

[0090] Several documents are cited throughout the text of thisspecification. Each of the documents cited herein (including anymanufacturer's specifications, instructions, etc.) are herebyincorporated herein by reference; however, there is no admission thatany document cited is indeed prior art as to the present invention.

[0091] The figures show:

[0092]FIG. 1. Proliferation of wild-type (circles) and reag-expressingCHO cells as a function of time. Cells were plated in 96-well dishes andat the indicated times the tetrazolium salt MTT⁶ (50 μg/nl) was added tothe plates. After four hours incubation in humidified atmosphere (37°C., 5%CO₂), the reaction was stopped by addition of 2 volumes of 10% SDSin 1M HCl. The blue formazan crystals produced in living cells weresolubilized overnight, and the resulting color was measured as opticaldensity at the indicated wavelength. Possible non-specific effects ofthe transfection on the cell proliferation can be neglected, since a)the results were comparable in three independent cell lines fromdifferent species (rat, hamster and human); b) transfection withdifferent independent clones gave the same results, and c) transfectionwith a different potassium channel (Kv1.4) in the same vector (thus witha tendency to recombine at the same site) gave results comparable to WTand did not reproduce the effects of the reag transfection.

[0093]FIG. 2. Proliferation of wild type (circles) and reag expressing(triangles) CHO cells, in the presence of 0.5% FCS. This serumconcentration is not able to sustain growth of normal cells, buttransfected cells complete almost three cycles. Methods as for FIG. 1.

[0094]FIG. 3. DNA synthesis in CHO cells expressing different potassiumchannels, in the presence of normal (10%) or low (0.5%) concentrationsof FCS. In control cells, WT or cells transfected with Kv1.4, the levelsof DNA synthesis drop significantly in the presence of low serumconcentration, whereas reag expressing cells maintain the samereplication levels as in high serum concentrations.

[0095]FIG. 4. (A) Photographs of plates with wild type, Kv1.4transfected or reag transfected NIH3T3 cells. The cells were seeded atlow density, and allowed to grow under standard conditions untilwild-type cells reached confluence. The cells were then fixed withmethanol and stained with Giernsa blue. Under those conditions, bothwild type and Kv1.4-expressing cells grow in a monolayer, whereas reagexpressing cells form foci (B) Foci formation of reag -transfectedNIH-3T3 cells compared to cells transfected with rKv1.4 and to wild typecells. The vector control (pcDNA3 transfected cells) yielded a similarphenotype as wild type cells (not shown). Transient transfection wascarried out using calcium phosphate (33). Cells were maintained in richmedium until control cells reached confluence, then fixed with methanoland stained with Giernsa blue.

[0096]FIG. 5. Currents elicited by depolarizations in MCF7 cells undervoltage clamp conditions. Left traces are whole cell currents, righttraces have been obtained in an excised outside-out patch. Both themacroscopic currents and the I-V relationships (C and D) are reminiscentof reag currents.

[0097]FIG. 6. Single channel activity in an outside-out membrane patchvoltage-clamped at 0 mV, in the presence or the absence of 5 μMastemizole. The pipette solution contained 140 mM KCl, 10 mM BAPTA, 10mM HEPES pH 7.2; the bath solution contained 140 mM NaCl, 2 mM CaCl₂, 2mM MgCl₂, 2.5 mM KCl, 10 HEPES pH 7.2.

[0098]FIG. 7. A. DNA synthesis in MCF7 cells under different eagblockers. B. HEK293 DNA synthesis levels in the presence of astemizole,glibenclamide and terfenadine.

[0099]FIG. 8. Dose-response curve for the effects of two H1 antagonistson DNA synthesis in MCF7 cells (IC50 7 and 10 mM for LY 91241 andastemizole respectively).

[0100]FIG. 9. Fluorescence images of control (untreated, A) andastemizole-treated (B) MCF7 cells, stained with Hoechst 33342. Notice inB the smaller surface of the nuclei, and a much lower cell density (dueto cell death).

[0101]FIG. 10. Nucleotide sequence of human-eag cDNA from human braincompared to the rat sequence and bovine sequences. Those positionsshowing a different nucleotide in any of the sequences are shaded.

[0102]FIG. 11. Amino acid sequences of both splice variants obtainedfrom human eag cDNA translation, compared to the corresponding bovine,mouse and rat sequences. The black boxes indicate a different residue inany of the sequences.

[0103]FIG. 12. RT-PCR from human brain, human mammary gland and MCF-7cells total RNA. The amplification produced two specific fragmentscorresponding to the expected sizes for heag 1 and 2 in brain, and theband corresponding to heag 1 in MCF-7 cells, while no amplification wasdetected in normal breast RNA.

[0104]FIG. 13. Voltage-dependence of activation in high extracellularpotassium, two-electrode voltage-clamp: In the conductance-voltage plot,the voltage for half-activation is shifted by 40 vM to the right in theheag channel with respect to the reag channel.

[0105]FIG. 14. Colony formation in semisolid medium of NIH-3T3 cellstransfected with the indicated DNAs. Cells were plated in regular mediumcontaining 0.3% agar onto a layer of 0.55% agar medium. Colonies largerthan 0.1 mm in diameter were scored 14 days after transfection. Theaverage number of colonies in at least ten counted microscope fields isexpressed per μg DNA used in the transfection (except for the lanes“Transfection buffer” and “No treatment”, where the numbers are absolutevalues). reag and Kv1.4 were transfected using either pcDNA3 or pTracerCMV vectors. FIG. 15. (A) Southern blot of RT-PCR products of RNAs fromdifferent human tissues and 293 cells. Transferrin receptor (TFR)signals are shown at the bottom. (B) Southern blot analysis of RT-PCRproducts of total RNAs from different human cell lines and mammaryepithelial cells in primary culture (Epith. cells). TRF signals areshown at the bottom.

[0106]FIG. 16. (A) Treatment of heag expressing tumor cell lines withantisense ODNs. (B) heag current in SHSY-5Y neuroblastoma cells (C)Current density in SHSY-5Y cells treated with antisense ODNs (D)Inhibition of DNA synthesis in human cancer cells (EFM-19, HeLa andSHSY-5Y) by antisense ODNs directed against heag.

[0107]FIG. 17. (A) Subcutaneous implantation of CHOHEAG cells inducedaggressive tumors that grew rapidly and soon broke the skin of thecarrier mice. The photograph was taken in the third weekpost-implantation of 2×10⁶ cells. (B.C) The average mass of CHOHEAGtumors was significantly greater than that of the CHOKv tumors both twoweeks (B; mean±S.E.M.; p=0.002) or three weeks post-implantation (C;mean±S.E.M.; p=0.03) (D) CHOHEAG and (E) CHOKv tumors photographed insitu. The main macroscopic differences are the darker color and thefixation to the skin of the CHOHEAG tumor. (F, G) CHOHEAG (F) and CHOKv(G) tumors were cut open to show the great extent of necrosis(arrowheads) in the former. (H, I) The greater degree of necrosis andthe fixation to the skin are also evident microscopically after paraffinembedding and hematoxylin-eosin staining. The histology is comparable inboth micrographs, but in (H) a much bigger necrotic area is observed(arrowheads), and there is no border between the subcutaneous fat andthe tumor. (Scale bars, 100 μm) (J) As a quantitative measurement ofthese images, the average width of the vital area in CHOKv tumors wassignificantly larger than that of CHOHEAG tumors (mean±S.E.M.;p<0.0005).

[0108]FIG. 18: Proliferation assays of rEAG-transfected CHO cells (A-C).Growth curves of CHO cells transfected with rEAG (circles) as comparedto naive cells (triangles) in 10% (filled symbols) or 0.5% (opensymbols) fetal calf serum. The values are referred to the ones measuredafter 12 h in culture (time 0 in the plot), and represent mean±S.E.M. ofeight wells in the same plate. Cell lines were established by selectionthrough the G-418 resistance encoded in the pcDNA3 vector. MTThydrolysis (22) was used to measure metabolic activity of viable cells.Serum was carefully diluted 12 hours after plating. (B) Increase inmetabolic activity during the first 12 hours after removal of S-phaseblock. For cell synchronization, 2 mM thymidine was added to the culturemedium for 12 h. Thymidine was removed from the medium for additional 12h, and then a second arresting pulse was applied for 12 h. Cells werethen trypsinized and plated for metabolic activity and DNA synthesisdetermination. (C) BrdU incorporation during the first 12 hours afterremoval of S-phase block for 12 h incubation in 10% FCS, or in thepresence of 0.5% FCS (24 h incubation). BrdU incorporation was measuredusing the Boehringer-Mannheim “BrdU labeling and detection kit”,following the indications of the manufacturer. The bars representmean±S.D. for wild-type CHO cells (open bars), Kv1.4-transfected (shadedbars) and eag-transfected (solid bars). The incorporation of BrdU isquantified as optical density at 405 n=(reference 490 nm) produced onABTS™ substrate by peroxidase coupled to the anti BrdU antibody.

[0109] The examples illustrate the invention.

EXAMPLE 1 Cloning of the K⁺ ion Channel

[0110] mRNA was purified from total RNA obtained from MCF-7 cellsfollowing standard procedures. Then, cDNA was prepared by reversetranscription with Superscript II reverse transcriptase; this cDNA wasused as a template for PCR amplification using degenerateoligonucleotides designed to match highly conserved eag sequences. Afteramplification, a SacII/SacII fragment from rat eag was used as a probefor Southern blot analysis of the results. Those bands showing positivehybridization were subsequently cloned in pGEM-T vector (Promega) andsequenced. All of them gave sequences corresponding to HERG.

[0111] Specific oligonucleotides engineered to avoid HERG cDNAamplification were then designed, taking into account rat, mouse andbovine eag. We looked for sequences having high homology between thevarious eag clones but with maximal divergence to the HERG sequence.

[0112] The sequences of the oligonucleotides were the following:5′-CAGAA(T,C)AA(T,C)GTGGC(A,C,T,G,)TGGCT 5′-TCACT(G,A)AAGATCTATA(A,G)TC

[0113] After PCR amplification, the band of the expected size was clonedinto pGEMT and sequenced. The sequence obtained showed high homology torat eag (nucleotides 942-1108).

[0114] This band was labeled and used as a probe to screen a mammarygland cDNA library. After screening of 2×10⁶ phages, no positive cloneswere found.

[0115] We then used specific oligonucleotides to analyze cDNA using PCRfrom human heart and human brain (obtained from total RNA purchased fromClontech). Two PCR products from brain were sequenced, and the sequencecorresponded to two alternatively spliced variants of eag. To furthertest the possibility of cloning the full length molecule from the humanbrain, we performed PCR analysis of a human cDNA library, and comparedthis result to the same experiment in the human mammary gland library(both from Clontech). Only the brain library gave positive results.

[0116] Subsequently, the amplified fragment was employed to screen thehuman brain library (2 rounds, 10⁶ phages) and several clones that werecloned into the pBSK-vector were found and sequenced. All of themcorresponded to the central part of the molecule, but were missing the5′ and 3′ ends. The longest of these positive clones was used to preparea probe and re-screen the library (again two rounds, 2×10⁶ clones).

[0117] The sequences obtained in this case corresponded to part of thecoding sequence (approximately 400 bp 5′ were missing until theinitiation codon) and a long 3′ untranslated sequence. Since thefragment close to the 5′ end of the molecule started in all cases withan EcoRI site, it was suspected that the site was actually present inthe heag sequence, and that is was lost in the subcloning of thefragments into vectors for sequencing.

[0118] To obtain the full length sequence, we pooled those phages thatcarried fragments close to the 5′ end and analyzed them by PCRamplification, using the sequence 3′ to the mentioned EcoRI site and asequence from lambda gt10 as primers for the PCR. After successivefractionation of the pools, two phages that carried the 5′ end of thecoding sequence were obtained, and one of them contained part of the 5′untranslated region.

[0119] Once we knew the complete sequence, we assembled the whole clonestarting from two phages, one of them containing the 3′ UTR and most ofthe coding sequence, and the other containing the 5′ end. The firstfragment was extracted from the phage by SphI/HindIII digestion, andsubdloned into pBKS- to produce pBKSheag 1. In this was, a 1.2 kbpSphI-SphI fragment was also removed from the clone, and it was necessaryto reintroduce it afterwards. The fragment containing the 5′ end wasextracted by HindIII/MunI digestion. This fragment was ligated with aHindIII/MunI digest of pBKSheag 1. Only using this procedure were weable to obtain the full length clone in a single plasmid. We then neededto reintroduce the SphI-SphI fragment since we had deleted one of theSphI sites. Subsequently, an EagI/NotI fragment was subcloned into theNotI site of pCDNA3 vector, to eliminate the contaminating phagesequences and to obtain a vector suitable for functional expression ofthe channel. Finally obtained sequences are depicted in sequence listingas SEQ ID No. 1 and SEQ ID No. 2.

EXAMPLE 2 Identification of Inhibitors that Specifically Bloc the Actionof Human Eag

[0120] Another member of the eag family, HERG¹¹⁻¹⁶, has been related toa familiar form of long QT syndrome (LQT). This has allowed to identifyseveral blockers of HERG based on their ability to induce LQT-typearrythmias. Thus, certain histamine H1 receptor blockers, such asastemizole and terfenadine, as well as class III antiarrythmic drugs(dofetilide, E-4031) are potent and specific blockers of HERG^(15,17).However, for eag channels, specific blockers have not yet beendescribed. Due to the sequence similarity between HERG and eag channels,both groups of drugs on reag were tested in accordance with the presentinvention. The H1 blockers also affect reag, whereas the channel israther insensitive to class III antiarrythmics (dofetilide). Thisprovides a useful tool to selectively block eag-type channels and todiscard possible effects of HERG channels (which are also present inMCF7 cells). The effect of one of these drugs (astemizole 5 μM) is shownon single putative human eag channels in FIG. 6.

[0121] It was further tested whether several reag and other potassiumchannel blockers are able to inhibit growth of MCF7 cells. As a“positive” control glibenclamide, a blocker of the ATP-sensitivepotassium channel was also included, since it has been described toinhibit the proliferation of this cell line². To determine the rate ofDNA synthesis, cells were plated on 96-well microtiter plates at adensity of ≈10⁵ cells/ml and in the absence of growth factors. After 24hours starvation, cells were stimulated by addition of 10% FCS in thepresence of BrdU. The amount of BrdU incorporated into the newlysynthesized DNA was determined using a commercial antibody (BoehringerMannheim). The drugs tested were added either at the same time or 12hours prior to the stimulation. In a different human cell line, HEK293,the addition of 10 μM astemizole or 100 μM glibenclamide did not reducesignificantly the DNA synthesis, while terfenadine (10 μM) produced astrong inhibition. For this reason, only effects of astemizole (and itsclosely related analog LY91241) were considered, and those produced byterfenadine (although MCF7 cells are significantly more sensitive togrowth inhibition by terfenadine than the control cells) discarded. InMCF7 cells, 5 μM astemizole reduced the DNA synthesis by 40%, while thesame concentration of the HERG-specific blocker dofetilide produced nosignificant effects. Ten times higher concentrations (50 μM) of otherpotassium channel blockers (quinidine or glibenclamide) where requiredto induce a similar effect. A dose-response curve for astemizole effectson DNA synthesis in MCF7 cells is depicted in FIG. 8. The half-maximaleffect was obtained for 10 μM astemizole.

[0122] In an attempt to clarify the mechanism underlying theproliferation inhibition in MCF7 cells, the nuclear morphology of cellstreated with 5 μM astemizole were checked, using the supravital nuclearstain Hoechst 33342. After 24 hours of treatment, most cells showednuclear condensation and fragmentation, typical features of apoptoticcell death (FIG. 9).

[0123] In conclusion, a human counterpart of the reag channels arepresent in human cancer cells, and they have the ability to inducemalignant transformation in several different cell types.

EXAMPLE 3 Expression of Heag in Different Human Tissues

[0124] 500 ng total RNA from different human tissues (or 5 ng polyA⁺RNA, for spinal chord) were reverse transcribed and amplified using apair of oligonucleotides of the sequences 5′-CGCATGAACTACCTGAAGACG(forward) and 5′-TCTGTGGATGGGGCGATGTTC (reverse). The amplified DNA wasanalyzed by Southern blot using a specific human eag probes (a 1.5 KbEcoRI fragment from the core of the channel). Among the RNAs tested,only brain total RNA gave positive signals. RNAs from spinal chord,adrenal gland, skeletal muscle, heart, trachea, liver, kidney andmammary gland were negative. The integrity of the RNA was checked usingtransferin amplification. Using the same approach, the expression ofheag in several tumoral human cell lines was checked, in: MCF-7 (breastadenocarcinoma), BT474 (breast ductal carcinoma, from a solid tumor),EFM-19 (breast carcinoma, ductal type, from pleural fluid), COLO-824(breast carcinoma, from pleural fluid), SHSY5Y (neuroblastoma).

[0125] In contrast to normal tissues, all the cancer cell lines testedwere found positive for heag expression.

[0126] Further, Southern blot of RT-PCR products of RNAs from differenthuman tissues and 293 cells show that only in RNA from brain the twobands corresponding to heag A and B could be amplified and identified.Transferrin receptor (TFR) signals are shown at the bottom (FIG. 15A).Furthermore, a Southern blot analysis of RT-PCR products of total RNAsfrom different human cell lines and mammary epithelial cells in primaryculture (Epith. cells). TRF signals are shown at the bottom. RNAs fromthe different cell lines (34) and commercial RNAs from human tissues(Clontech) were subjected to single-tube RT-PCR (35). Total RNA was usedwith the exception of spinal cord, where poly(A)⁺ RNA was used (Primersequences were: forward: 5′-CGCATGAACTACTGAAGACG, and reverse:5′-TCTGTGGATGGGGCGATGTTC. 5′-TCAGCCCAGCAGAAGCATTAT and reverse:5′-CTGGCAGCGTGTGAGAGC were used to control RNA and PCR performance.).Specific primers for TFR were used to control RNA and PCR performance.These ODNs were designed according to the published TFR sequence (36),starting at exon 11 and spanning to exon 19 (37). This, together withthe amplification of two heag splice fragments and controls in theabsence of reverse transcriptase, excludes a false positive due togenomic DNA contamination. 50 μl (heag) or 15 μl (TFR) of PCR reactionswere analyzed in 2% agarose gels. DNA was transferred to membranes andconsecutively hybridized at high stringency with [³²P]-dCTP labeledrandom primed probes consisting of a 980 bp heag fragment and the TFRfragment amplified from brain RNA.

EXAMPLE 4 Expression of heag in vivo

[0127] To determine whether the expression of heag is advantageous fortumor cells in vivo, the inventors preformed subcutaneous implants ofCHO cells expressing the channel (CHOHEAG cells) into the flank offemale scid (severe combined immunodeficiency, 33) mice. CHOKv cellswere used as a control. Therefore, 2×10⁶ CHOhEAG or CHO-Kv1.4 cellssuspended in 100 μl PBS were implanted subcutaneously on the flank of6-8 week old female Fox Chase scid mice (CB-17/Icr sicd/scid) obtainedfrom Bomholtgard, Ry, Denmark. The presence of tumors was checked everysecond day by tactile inspection of every mouse. After two or threeweeks, the animals were sacrificed by cervical dislocation and thetumors dissected and fixed in paraformaldehyde for subsequent paraffininclusion and staining. The identity of the CHOHEAG cells wasestablished by UV illumination of the tumors to evoke fluorescence fromthe green fluorescence protein encoded in the pTracer vector(Invitrogen). One week after the implantation, all CHOhEAG-injected micecarried tumors detectable by palpation, while no mass greater than 1 mmwas observed in the controls. During the second week post-implantation,the heag-expressing tumors reached in excess of 5 mm in diameter andvisibly emerged through the skin in most cases (FIG. 17A); the mice weresacrificed after two (N=6) or three weeks (N=7). Only one of the 11control animals used was free of visible tumors; all 13 CHOhEAG-injectedanimals showed tumors. The average mass (FIGS. 17B, C) of the heag-expressing tumors was significantly larger than that of controls,especially two weeks following implantation (FIG. 17B). From macroscopicobservation, the tumors appeared friable and hemorragic; the CHOHEAGtumors were darker than the controls and were adhered to the skin (FIGS.17D, E) in all CHOhEAG-injected mice at two weeks. Six of seven miceexhibited similar characteristics at three weeks. In contrast, the tumorcould be easily dissected from the skin inall of the control mice aftertwo weeks, and in five out of six mice at three weeks. The tissue belowthe tumor appeared unaffected in all cases. The dark color was due togreat extent of intratumoral necrosis (FIGS. 17F, G, arrows), confirmedby histology (FIGS. 17H, I, arrowheads), indicating a faster growth ofCHOhEAG tumors. The thickness of the vital area in the EAG-expressingtumors was significantly smaller than in the controls (FIG. 17J). Therapid growth of the tumor can account for the massive intratumoralnecrosis in the CHOHEAG group. This could also explain the enhanceddifference found in the mass of the tumors two weeks after implantation,since CHOhEAG tumors would cease growth due to massive necrosis. Thesedata strongly suggest that expression of heag tumors grow faster and aremore aggressive than CHOKv tumors.

EXAMPLE 5 Inhibition of heag

[0128] It is assumed that expression of heag in some tumor cells is notthe consequence of their abnormal growth, but that this K⁺ channel isnecessary for their proliferation. Therefore, inhibition of heagexpression with antisense oligodeoxynucleotides (ODNs) should decreasethe proliferation rate in these tumor cells. Therefore, a 19-merantisense phosphorothioate ODN (5′-CAGCCATGGTCATCCTCCC) spanning theputative initiation codon of heag was used to test inhibition ofproliferation. The sense ODN and a scrambled sequence(gtcggtaccagtaggaggg) were used as controls. Data shown in FIG. 16Aconfirms the efficiency of the antisense ODN treatment in reducing theheag mRNA content in EFM cells. A reduction in heag mediated K⁺ currentsin SHSY-5Y cells by treatment with antisense ODN is shown in FIGS. 16Band C.

[0129] Treatment of heag expressing tumor cell lines with antisense ODNssignificantly reduced the yield of amplified PCR products. EFM-19 cellswere treated with 10 μg/ml DAC30 (lanes “C”) or 10 μg/ml DAC30(Eurogentec) plus 1 μM antisense ODN (lanes “AS”) overnight, total RNAwas extracted and assayed under the same conditions as described inExample 3, with ODNs designed to either amplify heag or the transferrinreceptor. The arrows in FIG. 16A mark the expected sizes of theamplified fragments. Further, to dissect the heag current in SHSY-5Yneuroblastoma cells, the inventors utilized the voltage-dependence ofthe activation of eag (30) in the presence of extracellular Mg²⁺. Thecurrent was measured after a depolarization to +60 vM from −120 vM (FIG.16B, gray lines). The first part of the subtracted trace (FIG. 16B,black line) corresponds to eag current that has not yet activated whenthe holding potential is very negative (−120 vM), but becomes evident ifthe holding potential is −60 vM. The average current between 19 and 21ms was chosen to determine the current density. The current density inSHSY-5Y cells treated with antisense ODNs was significantly reduced ascompared to control cells (The electrophysiological determinations wereperformed using standard protocols in the whole cell configuration ofthe patch-clamp technique (Harill, O. P., Marty, A., Neher, E., Saklann,B., Sigworth, F. J. Pflügers Arch-Eur. J. Physiol 391, 85 (1981)), withan extracellular solution containing (mM) 140 NaCl, 2.5 KCl, 2 CaC12, 2MgC12, 10 Hepes/NaOH pH 7.2, 10 glucose. The pipette solution was (mM)140 KCl, 10 BAPTA, 10 Hepes/KOH pH 7.2.). The cells were treatedovernight with antisense ODN 1 μM containing fluorescein-labeled ODN.The currents were determined 1 to 3 days later in cells showingfluorescence in their nuclei. The bars in FIG. 16C represent mean±S.E.M.for 9 cells (control) or 25 cells (antisense). Only the outward currentswere evaluated in the analysis. Furthermore, the inhibition of DNAsynthesis in human cancer cells (EFM-19, HeLa and SHSY-5Y) by antisenseODNs directed against heag was investigated. DNA synthesis is expressedrelative to BrdU incorporation in the absence of ODNs. The uptakeconditions into cells using fluorescein labeled antisense ODN wasoptimized. Cells were seeded in 96-well plates at a density of 105cells/ml. One day after plating, the cells were washed with culturemedium and the ODN was added (final concentration 10 μM). The ODN hadpreviously been mixed with 20 μg/ml of the transfection ragenent DAC-30(Eurogentec) in serum-free medium and allowed to incubate at roomtemperature for 20-30 min. The mixture was then added as a 1:1 dilutionin culture medium and maintained in contact with cells overnight. Afterthis incubation, the cells were washed and labeled with BrdU (100 μM)for 2 h. Incorporation was detected using the kit from BoehringerMannheim and measured as OD units at 405 nm (reference 490 nm) aftersubtraction of the non-specific background incorporation. (FIG. 16D).The bars indicate mean±S.D. for eight wells per condition in arepresentative experiment.

Glossary and List of Abbreviations

[0130] Cell lines: CHO CHO-K1 (ATCC CCL 61) Chinese hamster Cricetulusgriseus ovary HEK293 293 (ATCC CRL 1573) Transformed primary humanembryonal kidney NIH3T3 (ATCC CRL 1658) Embryo Swiss mouse fibroblastsMCF7 (ATCC HTB 22) Human breast adenocarcinoma WT Wild-type cells

[0131] Genes and gene products eag ether-à-go-go potassium channel HERGHuman-Eag-Related Gene. Codes for an inwardly rectifying potassiumchannel mainly expressed in heart. Kv1.4 Inactivating voltage-dependentpotassium channel. Initially cloned from rat brain, it is present inmany other tissues.

[0132] Others EGF Epidermal growth factor PDGF Platelet-derived growthfactor FCS Fetal calf serum I-V relation Current-Voltage relation LQTLong Q-T (interval between Q and T waves in the electrocardiogram).Induces severe arrythmias due to repolarization defects. BrdU5-Bromo-2′-deoxyuridine. Structure analog of thymidine. IC50Concentration that produces 50% inhibition RT-PCR. Polymerase ChainReaction of cDNA produced by reverse transcription in the same tube.

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[0169] 37. Evans, P. and Kemp, J. Gene 199, 123 (1997).

[0170] This application incorporates by reference internationalapplication PCT/EP99/02695, filed Apr. 21, 1999, which designated theUnited States.

1 24 1 3002 DNA Homo sapiens 1 aattccgggc ccgccggacc ccgagctgctgggaggatga ccatggctgg gggcaggagg 60 ggactagtgg cccctcaaaa cacgtttctggagaatattg ttcggcggtc caatgatact 120 aattttgtgt tggggaatgc tcagatagtggactggccta ttgtgtacag caatgatgga 180 ttttgcaagc tgtctggcta tcacagggcagaagtgatgc aaaaaagcag cacctgcagt 240 tttatgtatg gggagctgac tgataaagacacgattgaaa aagtgcggca aacatttgag 300 aactatgaga tgaattcctt tgaaattctgatgtacaaga agaacaggac acctgtgtgg 360 ttctttgtga aaattgctcc aattcgaaacgaacaggata aagtggtttt atttctttgc 420 actttcagtg acataacagc tttcaaacagccaattgagg atgattcatg taaaggctgg 480 gggaagtttg ctcggctgac aagagcactgacaagcagca ggggtgtcct gcagcagctg 540 gctccaagcg tgcaaaaagg cgagaatgtccacaagcact cccgcctggc agaggtccta 600 cagctgggct cagacatcct tccccagtacaagcaagagg caccaaagac tccccctcac 660 atcatcttac attattgtgt ttttaagaccacgtgggatt ggatcatctt gatcttgacc 720 ttctatacag ccatcttggt cccttataatgtctccttca aaaccaggca gaataatgtg 780 gcctggctgg ttgttgatag catcgtggatgttatctttt tggtggacat tgtgctcaat 840 tttcatacca cctttgttgg accagcaggggaggtgattt ctgaccccaa acttatccgc 900 atgaactacc tgaagacgtg gtttgtgattgaccttctgt cctgtttgcc atatgatgtc 960 atcaacgctt ttgagaacgt ggatgagggcatcagcagcc tgttcagctc tctaaaagtt 1020 gtccggctgc tccgtcttgg gcgagtggcccgtaagctgg accactacat tgaatatgga 1080 gctgctgtgc tggtcctgct ggtgtgtgtgtttgggctgg ctgcacactg gatggcctgc 1140 atctggtaca gcattgggga ctatgagatctttgacgagg acaccaagac aatccgcaac 1200 aacagctggc tgtaccaact agcgatggacattggcaccc cttaccagtt taatgggtct 1260 ggctcaggga agtgggaagg tggtcccagcaagaattctg tctacatctc ctcgttgtat 1320 ttcacaatga ccagcctcac cagtgtgggctttgggaaca tcgccccatc cacagacatt 1380 gagaagatct ttgcagtggc catcatgatgattggctcac ttctctatgc caccatcttc 1440 gggaatgtga cgactatttt ccaacagatgtatgccaaca ccaacagata ccatgagatg 1500 ctcaacagtg ttcgggactt cctgaagctctaccaggtgc caaaaggatt gagtgagcga 1560 gtaatggatt atattgtgtc cacttggtccatgtccagag gcattgacac agagaaggtc 1620 ctgcagatct gccccaagga catgagagccgacatctgcg tgcacctgaa ccgcaaggtg 1680 ttcaaggagc acccggcctt ccggctggccagtgatggct gcctccgggc actggccatg 1740 gagttccaga cggtgcactg tgccccaggggacctcatct accatgcagg agagagcgtt 1800 gacagcctct gctttgtggt ttctggctccctggaggtga tccaagatga tgaggtggtg 1860 gccattctag gaaaaggaga cgtgtttggagatgtgttct ggaaggaagc cacccttgcc 1920 cagtcctgtg ccaatgttag ggccttgacctactgtgatc tgcatgtgat caagcgggat 1980 gccctgcaga aagtgctgga attctacacggccttctccc attccttctc ccggaacctg 2040 attctgacgt acaacttgag gaagaggattgtgttccgga agatcagcga tgtgaaacgt 2100 gaagaggaag aacgcatgaa acgaaagaatgaggcccccc tgatcttgcc cccggaccac 2160 cctgtccggc gcctcttcca gagattccgacagcagaaag aggccaggct ggcagctgag 2220 agagggggcc gggacctgga tgacctagatgtggagaagg gcaatgtcct tacagagcat 2280 gcctccgcca accacagcct cgtgaaggccagcgtggtca ccgtgcgtga gagtcctgcc 2340 acgcccgtat ccttccaggc agcctccacctccggggtgc cagaccacgc aaagctacag 2400 gcgccagggt ccgagtgcct gggccccaaggggggcgggg gcgattgtgc caagcgcaaa 2460 agctgggccc gcttcaaaga tgcttgcgggaagagtgagg actggaacaa ggtgtccaag 2520 gctgagtcga tggagacact tcccgagaggacaaaagcgt caggcgaggc cacactgaag 2580 aagacagact cgtgtgacag tggcatcaccaagagcgact tgcgcctgga caacgtgggt 2640 gaggccagga gtccccagga tcggagtcccatcctggcag aggtcaagca ttcgttctac 2700 cccatccctg agcagacgct gcaggccacagtcctggagg tgaggcacga gctgaaggag 2760 gacatcaagg ccttaaacgc caaaatgaccaatattgaga aacagctctc tgagatactc 2820 aggatattaa cttccagaag atcctctcagtctcctcagg agttgtttga aatatcgagg 2880 ccacagtccc cagaatcaga gagagacatttttggagcca gctgagaggt ctatttaaaa 2940 aaaaagtcag agacagatac ctccaaccctgccgtcacca ccacccctac cacccggaat 3000 tc 3002 2 3083 DNA Homo sapiens 2aattccgggc ccgccggacc ccgagctgct gggaggatga ccatggctgg gggcaggagg 60ggactagtgg cccctcaaaa cacgtttctg gagaatattg ttcggcggtc caatgatact 120aattttgtgt tggggaatgc tcagatagtg gactggccta ttgtgtacag caatgatgga 180ttttgcaagc tgtctggcta tcacagggca gaagtgatgc aaaaaagcag cacctgcagt 240tttatgtatg gggagctgac tgataaagac acgattgaaa aagtgcggca aacatttgag 300aactatgaga tgaattcctt tgaaattctg atgtacaaga agaacaggac acctgtgtgg 360ttctttgtga aaattgctcc aattcgaaac gaacaggata aagtggtttt atttctttgc 420actttcagtg acataacagc tttcaaacag ccaattgagg atgattcatg taaaggctgg 480gggaagtttg ctcggctgac aagagcactg acaagcagca ggggtgtcct gcagcagctg 540gctccaagcg tgcaaaaagg cgagaatgtc cacaagcact cccgcctggc agaggtccta 600cagctgggct cagacatcct tccccagtac aagcaagagg caccaaagac tccccctcac 660atcatcttac attattgtgt ttttaagacc acgtgggatt ggatcatctt gatcttgacc 720ttctatacag ccatcttggt cccttataat gtctccttca aaaccaggca gaataatgtg 780gcctggctgg ttgttgatag catcgtggat gttatctttt tggtggacat tgtgctcaat 840tttcatacca cctttgttgg accagcaggg gaggtgattt ctgaccccaa acttatccgc 900atgaactacc tgaagacgtg gtttgtgatt gaccttctgt cctgtttgcc atatgatgtc 960atcaacgctt ttgagaacgt ggatgaggtt agtgccttta tgggtgatcc agggaagatt 1020ggttttgctg atcagattcc accaccactg gaggggagag agagtcaggg catcagcagc 1080ctgttcagct ctctaaaagt tgtccggctg ctccgtcttg ggcgagtggc ccgtaagctg 1140gaccactaca ttgaatatgg agctgctgtg ctggtcctgc tggtgtgtgt gtttgggctg 1200gctgcacact ggatggcctg catctggtac agcattgggg actatgagat ctttgacgag 1260gacaccaaga caatccgcaa caacagctgg ctgtaccaac tagcgatgga cattggcacc 1320ccttaccagt ttaatgggtc tggctcaggg aagtgggaag gtggtcccag caagaattct 1380gtctacatct cctcgttgta tttcacaatg accagcctca ccagtgtggg ctttgggaac 1440atcgccccat ccacagacat tgagaagatc tttgcagtgg ccatcatgat gattggctca 1500cttctctatg ccaccatctt cgggaatgtg acgactattt tccaacagat gtatgccaac 1560accaacagat accatgagat gctcaacagt gttcgggact tcctgaagct ctaccaggtg 1620ccaaaaggat tgagtgagcg agtaatggat tatattgtgt ccacttggtc catgtccaga 1680ggcattgaca cagagaaggt cctgcagatc tgccccaagg acatgagagc cgacatctgc 1740gtgcacctga accgcaaggt gttcaaggag cacccggcct tccggctggc cagtgatggc 1800tgcctccggg cactggccat ggagttccag acggtgcact gtgccccagg ggacctcatc 1860taccatgcag gagagagcgt tgacagcctc tgctttgtgg tttctggctc cctggaggtg 1920atccaagatg atgaggtggt ggccattcta ggaaaaggag acgtgtttgg agatgtgttc 1980tggaaggaag ccacccttgc ccagtcctgt gccaatgtta gggccttgac ctactgtgat 2040ctgcatgtga tcaagcggga tgccctgcag aaagtgctgg aattctacac ggccttctcc 2100cattccttct cccggaacct gattctgacg tacaacttga ggaagaggat tgtgttccgg 2160aagatcagcg atgtgaaacg tgaagaggaa gaacgcatga aacgaaagaa tgaggccccc 2220ctgatcttgc ccccggacca ccctgtccgg cgcctcttcc agagattccg acagcagaaa 2280gaggccaggc tggcagctga gagagggggc cgggacctgg atgacctaga tgtggagaag 2340ggcaatgtcc ttacagagca tgcctccgcc aaccacagcc tcgtgaaggc cagcgtggtc 2400accgtgcgtg agagtcctgc cacgcccgta tccttccagg cagcctccac ctccggggtg 2460ccagaccacg caaagctaca ggcgccaggg tccgagtgcc tgggccccaa ggggggcggg 2520ggcgattgtg ccaagcgcaa aagctgggcc cgcttcaaag atgcttgcgg gaagagtgag 2580gactggaaca aggtgtccaa ggctgagtcg atggagacac ttcccgagag gacaaaagcg 2640tcaggcgagg ccacactgaa gaagacagac tcgtgtgaca gtggcatcac caagagcgac 2700ttgcgcctgg acaacgtggg tgaggccagg agtccccagg atcggagtcc catcctggca 2760gaggtcaagc attcgttcta ccccatccct gagcagacgc tgcaggccac agtcctggag 2820gtgaggcacg agctgaagga ggacatcaag gccttaaacg ccaaaatgac caatattgag 2880aaacagctct ctgagatact caggatatta acttccagaa gatcctctca gtctcctcag 2940gagttgtttg aaatatcgag gccacagtcc ccagaatcag agagagacat ttttggagcc 3000agctgagagg tctatttaaa aaaaaagtca gagacagata cctccaaccc tgccgtcacc 3060accaccccta ccacccggaa ttc 3083 3 962 PRT Homo sapiens 3 Met Thr Met AlaGly Gly Arg Arg Gly Leu Val Ala Pro Gln Asn Thr 1 5 10 15 Phe Leu GluAsn Ile Val Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 20 25 30 Gly Asn AlaGln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly 35 40 45 Phe Cys LysLeu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 50 55 60 Ser Thr CysSer Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Ile 65 70 75 80 Glu LysVal Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu 85 90 95 Ile LeuMet Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Phe Val Lys 100 105 110 IleAla Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe Leu Cys 115 120 125Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu Asp Asp Ser 130 135140 Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg Ala Leu Thr Ser 145150 155 160 Ser Arg Gly Val Leu Gln Gln Leu Ala Pro Ser Val Gln Lys GlyGlu 165 170 175 Asn Val His Lys His Ser Arg Leu Ala Glu Val Leu Gln LeuGly Ser 180 185 190 Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro Lys ThrPro Pro His 195 200 205 Ile Ile Leu His Tyr Cys Val Phe Lys Thr Thr TrpAsp Trp Ile Ile 210 215 220 Leu Ile Leu Thr Phe Tyr Thr Ala Ile Leu ValPro Tyr Asn Val Ser 225 230 235 240 Phe Lys Thr Arg Gln Asn Asn Val AlaTrp Leu Val Val Asp Ser Ile 245 250 255 Val Asp Val Ile Phe Leu Val AspIle Val Leu Asn Phe His Thr Thr 260 265 270 Phe Val Gly Pro Ala Gly GluVal Ile Ser Asp Pro Lys Leu Ile Arg 275 280 285 Met Asn Tyr Leu Lys ThrTrp Phe Val Ile Asp Leu Leu Ser Cys Leu 290 295 300 Pro Tyr Asp Val IleAsn Ala Phe Glu Asn Val Asp Glu Gly Ile Ser 305 310 315 320 Ser Leu PheSer Ser Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg 325 330 335 Val AlaArg Lys Leu Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu 340 345 350 ValLeu Leu Val Cys Val Phe Gly Leu Ala Ala His Trp Met Ala Cys 355 360 365Ile Trp Tyr Ser Ile Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr Lys 370 375380 Thr Ile Arg Asn Asn Ser Trp Leu Tyr Gln Leu Ala Met Asp Ile Gly 385390 395 400 Thr Pro Tyr Gln Phe Asn Gly Ser Gly Ser Gly Lys Trp Glu GlyGly 405 410 415 Pro Ser Lys Asn Ser Val Tyr Ile Ser Ser Leu Tyr Phe ThrMet Thr 420 425 430 Ser Leu Thr Ser Val Gly Phe Gly Asn Ile Ala Pro SerThr Asp Ile 435 440 445 Glu Lys Ile Phe Ala Val Ala Ile Met Met Ile GlySer Leu Leu Tyr 450 455 460 Ala Thr Ile Phe Gly Asn Val Thr Thr Ile PheGln Gln Met Tyr Ala 465 470 475 480 Asn Thr Asn Arg Tyr His Glu Met LeuAsn Ser Val Arg Asp Phe Leu 485 490 495 Lys Leu Tyr Gln Val Pro Lys GlyLeu Ser Glu Arg Val Met Asp Tyr 500 505 510 Ile Val Ser Thr Trp Ser MetSer Arg Gly Ile Asp Thr Glu Lys Val 515 520 525 Leu Gln Ile Cys Pro LysAsp Met Arg Ala Asp Ile Cys Val His Leu 530 535 540 Asn Arg Lys Val PheLys Glu His Pro Ala Phe Arg Leu Ala Ser Asp 545 550 555 560 Gly Cys LeuArg Ala Leu Ala Met Glu Phe Gln Thr Val His Cys Ala 565 570 575 Pro GlyAsp Leu Ile Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys 580 585 590 PheVal Val Ser Gly Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val 595 600 605Ala Ile Leu Gly Lys Gly Asp Val Phe Gly Asp Val Phe Trp Lys Glu 610 615620 Ala Thr Leu Ala Gln Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr Cys 625630 635 640 Asp Leu His Val Ile Lys Arg Asp Ala Leu Gln Lys Val Leu GluPhe 645 650 655 Tyr Thr Ala Phe Ser His Ser Phe Ser Arg Asn Leu Ile LeuThr Tyr 660 665 670 Asn Leu Arg Lys Arg Ile Val Phe Arg Lys Ile Ser AspVal Lys Arg 675 680 685 Glu Glu Glu Glu Arg Met Lys Arg Lys Asn Glu AlaPro Leu Ile Leu 690 695 700 Pro Pro Asp His Pro Val Arg Arg Leu Phe GlnArg Phe Arg Gln Gln 705 710 715 720 Lys Glu Ala Arg Leu Ala Ala Glu ArgGly Gly Arg Asp Leu Asp Asp 725 730 735 Leu Asp Val Glu Lys Gly Asn ValLeu Thr Glu His Ala Ser Ala Asn 740 745 750 His Ser Leu Val Lys Ala SerVal Val Thr Val Arg Glu Ser Pro Ala 755 760 765 Thr Pro Val Ser Phe GlnAla Ala Ser Thr Ser Gly Val Pro Asp His 770 775 780 Ala Lys Leu Gln AlaPro Gly Ser Glu Cys Leu Gly Pro Lys Gly Gly 785 790 795 800 Gly Gly AspCys Ala Lys Arg Lys Ser Trp Ala Arg Phe Lys Asp Ala 805 810 815 Cys GlyLys Ser Glu Asp Trp Asn Lys Val Ser Lys Ala Glu Ser Met 820 825 830 GluThr Leu Pro Glu Arg Thr Lys Ala Ser Gly Glu Ala Thr Leu Lys 835 840 845Lys Thr Asp Ser Cys Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu 850 855860 Asp Asn Val Gly Glu Ala Arg Ser Pro Gln Asp Arg Ser Pro Ile Leu 865870 875 880 Ala Glu Val Lys His Ser Phe Tyr Pro Ile Pro Glu Gln Thr LeuGln 885 890 895 Ala Thr Val Leu Glu Val Arg His Glu Leu Lys Glu Asp IleLys Ala 900 905 910 Leu Asn Ala Lys Met Thr Asn Ile Glu Lys Gln Leu SerGlu Ile Leu 915 920 925 Arg Ile Leu Thr Ser Arg Arg Ser Ser Gln Ser ProGln Glu Leu Phe 930 935 940 Glu Ile Ser Arg Pro Gln Ser Pro Glu Ser GluArg Asp Ile Phe Gly 945 950 955 960 Ala Ser 4 989 PRT Homo sapiens 4 MetThr Met Ala Gly Gly Arg Arg Gly Leu Val Ala Pro Gln Asn Thr 1 5 10 15Phe Leu Glu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 20 25 30Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly 35 40 45Phe Cys Lys Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 50 55 60Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Ile 65 70 7580 Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu 85 9095 Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Phe Val Lys 100105 110 Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe Leu Cys115 120 125 Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu Asp AspSer 130 135 140 Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg Ala LeuThr Ser 145 150 155 160 Ser Arg Gly Val Leu Gln Gln Leu Ala Pro Ser ValGln Lys Gly Glu 165 170 175 Asn Val His Lys His Ser Arg Leu Ala Glu ValLeu Gln Leu Gly Ser 180 185 190 Asp Ile Leu Pro Gln Tyr Lys Gln Glu AlaPro Lys Thr Pro Pro His 195 200 205 Ile Ile Leu His Tyr Cys Val Phe LysThr Thr Trp Asp Trp Ile Ile 210 215 220 Leu Ile Leu Thr Phe Tyr Thr AlaIle Leu Val Pro Tyr Asn Val Ser 225 230 235 240 Phe Lys Thr Arg Gln AsnAsn Val Ala Trp Leu Val Val Asp Ser Ile 245 250 255 Val Asp Val Ile PheLeu Val Asp Ile Val Leu Asn Phe His Thr Thr 260 265 270 Phe Val Gly ProAla Gly Glu Val Ile Ser Asp Pro Lys Leu Ile Arg 275 280 285 Met Asn TyrLeu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290 295 300 Pro TyrAsp Val Ile Asn Ala Phe Glu Asn Val Asp Glu Val Ser Ala 305 310 315 320Phe Met Gly Asp Pro Gly Lys Ile Gly Phe Ala Asp Gln Ile Pro Pro 325 330335 Pro Leu Glu Gly Arg Glu Ser Gln Gly Ile Ser Ser Leu Phe Ser Ser 340345 350 Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg Val Ala Arg Lys Leu355 360 365 Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu Val Leu Leu ValCys 370 375 380 Val Phe Gly Leu Ala Ala His Trp Met Ala Cys Ile Trp TyrSer Ile 385 390 395 400 Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr Lys ThrIle Arg Asn Asn 405 410 415 Ser Trp Leu Tyr Gln Leu Ala Met Asp Ile GlyThr Pro Tyr Gln Phe 420 425 430 Asn Gly Ser Gly Ser Gly Lys Trp Glu GlyGly Pro Ser Lys Asn Ser 435 440 445 Val Tyr Ile Ser Ser Leu Tyr Phe ThrMet Thr Ser Leu Thr Ser Val 450 455 460 Gly Phe Gly Asn Ile Ala Pro SerThr Asp Ile Glu Lys Ile Phe Ala 465 470 475 480 Val Ala Ile Met Met IleGly Ser Leu Leu Tyr Ala Thr Ile Phe Gly 485 490 495 Asn Val Thr Thr IlePhe Gln Gln Met Tyr Ala Asn Thr Asn Arg Tyr 500 505 510 His Glu Met LeuAsn Ser Val Arg Asp Phe Leu Lys Leu Tyr Gln Val 515 520 525 Pro Lys GlyLeu Ser Glu Arg Val Met Asp Tyr Ile Val Ser Thr Trp 530 535 540 Ser MetSer Arg Gly Ile Asp Thr Glu Lys Val Leu Gln Ile Cys Pro 545 550 555 560Lys Asp Met Arg Ala Asp Ile Cys Val His Leu Asn Arg Lys Val Phe 565 570575 Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp Gly Cys Leu Arg Ala 580585 590 Leu Ala Met Glu Phe Gln Thr Val His Cys Ala Pro Gly Asp Leu Ile595 600 605 Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys Phe Val Val SerGly 610 615 620 Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val Ala Ile LeuGly Lys 625 630 635 640 Gly Asp Val Phe Gly Asp Val Phe Trp Lys Glu AlaThr Leu Ala Gln 645 650 655 Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr CysAsp Leu His Val Ile 660 665 670 Lys Arg Asp Ala Leu Gln Lys Val Leu GluPhe Tyr Thr Ala Phe Ser 675 680 685 His Ser Phe Ser Arg Asn Leu Ile LeuThr Tyr Asn Leu Arg Lys Arg 690 695 700 Ile Val Phe Arg Lys Ile Ser AspVal Lys Arg Glu Glu Glu Glu Arg 705 710 715 720 Met Lys Arg Lys Asn GluAla Pro Leu Ile Leu Pro Pro Asp His Pro 725 730 735 Val Arg Arg Leu PheGln Arg Phe Arg Gln Gln Lys Glu Ala Arg Leu 740 745 750 Ala Ala Glu ArgGly Gly Arg Asp Leu Asp Asp Leu Asp Val Glu Lys 755 760 765 Gly Asn ValLeu Thr Glu His Ala Ser Ala Asn His Ser Leu Val Lys 770 775 780 Ala SerVal Val Thr Val Arg Glu Ser Pro Ala Thr Pro Val Ser Phe 785 790 795 800Gln Ala Ala Ser Thr Ser Gly Val Pro Asp His Ala Lys Leu Gln Ala 805 810815 Pro Gly Ser Glu Cys Leu Gly Pro Lys Gly Gly Gly Gly Asp Cys Ala 820825 830 Lys Arg Lys Ser Trp Ala Arg Phe Lys Asp Ala Cys Gly Lys Ser Glu835 840 845 Asp Trp Asn Lys Val Ser Lys Ala Glu Ser Met Glu Thr Leu ProGlu 850 855 860 Arg Thr Lys Ala Ser Gly Glu Ala Thr Leu Lys Lys Thr AspSer Cys 865 870 875 880 Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu AspAsn Val Gly Glu 885 890 895 Ala Arg Ser Pro Gln Asp Arg Ser Pro Ile LeuAla Glu Val Lys His 900 905 910 Ser Phe Tyr Pro Ile Pro Glu Gln Thr LeuGln Ala Thr Val Leu Glu 915 920 925 Val Arg His Glu Leu Lys Glu Asp IleLys Ala Leu Asn Ala Lys Met 930 935 940 Thr Asn Ile Glu Lys Gln Leu SerGlu Ile Leu Arg Ile Leu Thr Ser 945 950 955 960 Arg Arg Ser Ser Gln SerPro Gln Glu Leu Phe Glu Ile Ser Arg Pro 965 970 975 Gln Ser Pro Glu SerGlu Arg Asp Ile Phe Gly Ala Ser 980 985 5 18 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 5 ccaaacacac acaccagc18 6 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 6 cgtggatgtt atctttttgg 20 7 18 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 7 gggaggatga ccatggct18 8 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 8 cagaayaayg tggcntggct 20 9 19 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 9 tcactraaga tctatartc19 10 21 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 10 cgcatgaact acctgaagac g 21 11 21 DNA ArtificialSequence Description of Artificial Sequence Synthetic DNA 11 tctgtggatggggcgatgtt c 21 12 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 12 gggaggatga ccatggct 18 13 2886 DNA Homosapiens 13 atgaccatgg ctgggggcag gaggggacta gtggcccctc aaaacacgtttctggagaat 60 attgttcggc ggtccaatga tactaatttt gtgttgggga atgctcagatagtggactgg 120 cctattgtgt acagcaatga tggattttgc aagctgtctg gctatcacagggcagaagtg 180 atgcaaaaaa gcagcacctg cagttttatg tatggggagc tgactgataaagacacgatt 240 gaaaaagtgc ggcaaacatt tgagaactat gagatgaatt cctttgaaattctgatgtac 300 aagaagaaca ggacacctgt gtggttcttt gtgaaaattg ctccaattcgaaacgaacag 360 gataaagtgg ttttatttct ttgcactttc agtgacataa cagctttcaaacagccaatt 420 gaggatgatt catgtaaagg ctgggggaag tttgctcggc tgacaagagcactgacaagc 480 agcaggggtg tcctgcagca gctggctcca agcgtgcaaa aaggcgagaatgtccacaag 540 cactcccgcc tggcagaggt cctacagctg ggctcagaca tccttccccagtacaagcaa 600 gaggcaccaa agactccccc tcacatcatc ttacattatt gtgtttttaagaccacgtgg 660 gattggatca tcttgatctt gaccttctat acagccatct tggtcccttataatgtctcc 720 ttcaaaacca ggcagaataa tgtggcctgg ctggttgttg atagcatcgtggatgttatc 780 tttttggtgg acattgtgct caattttcat accacctttg ttggaccagcaggggaggtg 840 atttctgacc ccaaacttat ccgcatgaac tacctgaaga cgtggtttgtgattgacctt 900 ctgtcctgtt tgccatatga tgtcatcaac gcttttgaga acgtggatgagggcatcagc 960 agcctgttca gctctctaaa agttgtccgg ctgctccgtc ttgggcgagtggcccgtaag 1020 ctggaccact acattgaata tggagctgct gtgctggtcc tgctggtgtgtgtgtttggg 1080 ctggctgcac actggatggc ctgcatctgg tacagcattg gggactatgagatctttgac 1140 gaggacacca agacaatccg caacaacagc tggctgtacc aactagcgatggacattggc 1200 accccttacc agtttaatgg gtctggctca gggaagtggg aaggtggtcccagcaagaat 1260 tctgtctaca tctcctcgtt gtatttcaca atgaccagcc tcaccagtgtgggctttggg 1320 aacatcgccc catccacaga cattgagaag atctttgcag tggccatcatgatgattggc 1380 tcacttctct atgccaccat cttcgggaat gtgacgacta ttttccaacagatgtatgcc 1440 aacaccaaca gataccatga gatgctcaac agtgttcggg acttcctgaagctctaccag 1500 gtgccaaaag gattgagtga gcgagtaatg gattatattg tgtccacttggtccatgtcc 1560 agaggcattg acacagagaa ggtcctgcag atctgcccca aggacatgagagccgacatc 1620 tgcgtgcacc tgaaccgcaa ggtgttcaag gagcacccgg ccttccggctggccagtgat 1680 ggctgcctcc gggcactggc catggagttc cagacggtgc actgtgccccaggggacctc 1740 atctaccatg caggagagag cgttgacagc ctctgctttg tggtttctggctccctggag 1800 gtgatccaag atgatgaggt ggtggccatt ctaggaaaag gagacgtgtttggagatgtg 1860 ttctggaagg aagccaccct tgcccagtcc tgtgccaatg ttagggccttgacctactgt 1920 gatctgcatg tgatcaagcg ggatgccctg cagaaagtgc tggaattctacacggccttc 1980 tcccattcct tctcccggaa cctgattctg acgtacaact tgaggaagaggattgtgttc 2040 cggaagatca gcgatgtgaa acgtgaagag gaagaacgca tgaaacgaaagaatgaggcc 2100 cccctgatct tgcccccgga ccaccctgtc cggcgcctct tccagagattccgacagcag 2160 aaagaggcca ggctggcagc tgagagaggg ggccgggacc tggatgacctagatgtggag 2220 aagggcaatg tccttacaga gcatgcctcc gccaaccaca gcctcgtgaaggccagcgtg 2280 gtcaccgtgc gtgagagtcc tgccacgccc gtatccttcc aggcagcctccacctccggg 2340 gtgccagacc acgcaaagct acaggcgcca gggtccgagt gcctgggccccaaggggggc 2400 gggggcgatt gtgccaagcg caaaagctgg gcccgcttca aagatgcttgcgggaagagt 2460 gaggactgga acaaggtgtc caaggctgag tcgatggaga cacttcccgagaggacaaaa 2520 gcgtcaggcg aggccacact gaagaagaca gactcgtgtg acagtggcatcaccaagagc 2580 gacttgcgcc tggacaacgt gggtgaggcc aggagtcccc aggatcggagtcccatcctg 2640 gcagaggtca agcattcgtt ctaccccatc cctgagcaga cgctgcaggccacagtcctg 2700 gaggtgaggc acgagctgaa ggaggacatc aaggccttaa acgccaaaatgaccaatatt 2760 gagaaacagc tctctgagat actcaggata ttaacttcca gaagatcctctcagtctcct 2820 caggagttgt ttgaaatatc gaggccacag tccccagaat cagagagagacatttttgga 2880 gccagc 2886 14 2967 DNA Homo sapiens 14 atgaccatggctgggggcag gaggggacta gtggcccctc aaaacacgtt tctggagaat 60 attgttcggcggtccaatga tactaatttt gtgttgggga atgctcagat agtggactgg 120 cctattgtgtacagcaatga tggattttgc aagctgtctg gctatcacag ggcagaagtg 180 atgcaaaaaagcagcacctg cagttttatg tatggggagc tgactgataa agacacgatt 240 gaaaaagtgcggcaaacatt tgagaactat gagatgaatt cctttgaaat tctgatgtac 300 aagaagaacaggacacctgt gtggttcttt gtgaaaattg ctccaattcg aaacgaacag 360 gataaagtggttttatttct ttgcactttc agtgacataa cagctttcaa acagccaatt 420 gaggatgattcatgtaaagg ctgggggaag tttgctcggc tgacaagagc actgacaagc 480 agcaggggtgtcctgcagca gctggctcca agcgtgcaaa aaggcgagaa tgtccacaag 540 cactcccgcctggcagaggt cctacagctg ggctcagaca tccttcccca gtacaagcaa 600 gaggcaccaaagactccccc tcacatcatc ttacattatt gtgtttttaa gaccacgtgg 660 gattggatcatcttgatctt gaccttctat acagccatct tggtccctta taatgtctcc 720 ttcaaaaccaggcagaataa tgtggcctgg ctggttgttg atagcatcgt ggatgttatc 780 tttttggtggacattgtgct caattttcat accacctttg ttggaccagc aggggaggtg 840 atttctgaccccaaacttat ccgcatgaac tacctgaaga cgtggtttgt gattgacctt 900 ctgtcctgtttgccatatga tgtcatcaac gcttttgaga acgtggatga ggttagtgcc 960 tttatgggtgatccagggaa gattggtttt gctgatcaga ttccaccacc actggagggg 1020 agagagagtcagggcatcag cagcctgttc agctctctaa aagttgtccg gctgctccgt 1080 cttgggcgagtggcccgtaa gctggaccac tacattgaat atggagctgc tgtgctggtc 1140 ctgctggtgtgtgtgtttgg gctggctgca cactggatgg cctgcatctg gtacagcatt 1200 ggggactatgagatctttga cgaggacacc aagacaatcc gcaacaacag ctggctgtac 1260 caactagcgatggacattgg caccccttac cagtttaatg ggtctggctc agggaagtgg 1320 gaaggtggtcccagcaagaa ttctgtctac atctcctcgt tgtatttcac aatgaccagc 1380 ctcaccagtgtgggctttgg gaacatcgcc ccatccacag acattgagaa gatctttgca 1440 gtggccatcatgatgattgg ctcacttctc tatgccacca tcttcgggaa tgtgacgact 1500 attttccaacagatgtatgc caacaccaac agataccatg agatgctcaa cagtgttcgg 1560 gacttcctgaagctctacca ggtgccaaaa ggattgagtg agcgagtaat ggattatatt 1620 gtgtccacttggtccatgtc cagaggcatt gacacagaga aggtcctgca gatctgcccc 1680 aaggacatgagagccgacat ctgcgtgcac ctgaaccgca aggtgttcaa ggagcacccg 1740 gccttccggctggccagtga tggctgcctc cgggcactgg ccatggagtt ccagacggtg 1800 cactgtgccccaggggacct catctaccat gcaggagaga gcgttgacag cctctgcttt 1860 gtggtttctggctccctgga ggtgatccaa gatgatgagg tggtggccat tctaggaaaa 1920 ggagacgtgtttggagatgt gttctggaag gaagccaccc ttgcccagtc ctgtgccaat 1980 gttagggccttgacctactg tgatctgcat gtgatcaagc gggatgccct gcagaaagtg 2040 ctggaattctacacggcctt ctcccattcc ttctcccgga acctgattct gacgtacaac 2100 ttgaggaagaggattgtgtt ccggaagatc agcgatgtga aacgtgaaga ggaagaacgc 2160 atgaaacgaaagaatgaggc ccccctgatc ttgcccccgg accaccctgt ccggcgcctc 2220 ttccagagattccgacagca gaaagaggcc aggctggcag ctgagagagg gggccgggac 2280 ctggatgacctagatgtgga gaagggcaat gtccttacag agcatgcctc cgccaaccac 2340 agcctcgtgaaggccagcgt ggtcaccgtg cgtgagagtc ctgccacgcc cgtatccttc 2400 caggcagcctccacctccgg ggtgccagac cacgcaaagc tacaggcgcc agggtccgag 2460 tgcctgggccccaagggggg cgggggcgat tgtgccaagc gcaaaagctg ggcccgcttc 2520 aaagatgcttgcgggaagag tgaggactgg aacaaggtgt ccaaggctga gtcgatggag 2580 acacttcccgagaggacaaa agcgtcaggc gaggccacac tgaagaagac agactcgtgt 2640 gacagtggcatcaccaagag cgacttgcgc ctggacaacg tgggtgaggc caggagtccc 2700 caggatcggagtcccatcct ggcagaggtc aagcattcgt tctaccccat ccctgagcag 2760 acgctgcaggccacagtcct ggaggtgagg cacgagctga aggaggacat caaggcctta 2820 aacgccaaaatgaccaatat tgagaaacag ctctctgaga tactcaggat attaacttcc 2880 agaagatcctctcagtctcc tcaggagttg tttgaaatat cgaggccaca gtccccagaa 2940 tcagagagagacatttttgg agccagc 2967 15 19 DNA Artificial Sequence Description ofArtificial Sequence Antisense phosphorothioate ODN 15 cagccatggtcatcctccc 19 16 19 DNA Artificial Sequence Description of ArtificialSequence Synthetic scrambled sequence 16 gtcggtacca gtaggaggg 19 17 21DNA Artificial Sequence Description of Artificial Sequence Primer 17tcagcccagc agaagcatta t 21 18 18 DNA Artificial Sequence Description ofArtificial Sequence Primer 18 ctggcagcgt gtgagagc 18 19 3041 DNA Bovinesp. 19 gtgccgggac gccccccaga ccccgagctg ccgggaggat gaccatggct gggggcagga60 agggactggt ggccccgcaa aacacgtttc tggagaatat tgtccggcgg tccaatgata 120ctaactttgt tttggggaat gcccagatag tggactggcc tatcgtgtac agcaatgatg 180gattttgcaa gctgtctggc tatcacaggg cggaagtgat gcaaaaaagc agtacatgca 240gttttatgta tggggagctg accgataaag ataccattga aaaagtgcgg caaacctttg 300agaactatga gatgaattcc tttgaaattc tgatgtacaa gaagaacagg acacctgtgt 360ggttctttgt gaaaattgct ccaattcgaa acgaacagga taaagtggtt ttatttcttt 420gcactttcag tgacataacc gctttcaaac agccgattga agatgattca tgtaaaggct 480gggggaagtt cgctcggctg accagagcac tgacgagcag ccggggtgtc ctgcagcagc 540tggctcccag cgtgcagaaa ggcgagaacg tccacaagca ctcccgtctg gccgaggttc 600tgcagctggg ctcagacatc cttccccagt acaagcaaga ggcaccaaag actcccccgc 660acatcatctt acactactgc gtttttaaga ccacgtggga ctggatcatc ctgatcctaa 720ccttctacac agccatcctg gttccttaca acgtctcctt taaaaccagg cagaacaacg 780tggcctggct ggttgtggac agcatcgtgg atgtcatttt tttggtggac attgtgctga 840attttcacac cacttttgtt ggacccgctg gggaggtgat ttctgacccc aaactcattc 900gcatgaacta cctgaagacg tggtttgtga ttgaccttct gtcctgtttg ccctatgacg 960tcatcaacgc ttttgagaac gtggatgagg gcatcagcag cctgttcagc tctctgaaag 1020ttgtccggct gctccgcctg ggacgcgtgg cccggaagct ggaccactac atcgagtatg 1080gagctgccgt gctggtcctg ctggtgtgtg tgttcgggct ggccgctcac tggatggcct 1140gcatttggta cagcatcggg gactatgaga tcttcgacga ggacaccaag accatccgca 1200acaacagctg gctctaccag ctggccatgg acattggcac cccttaccag tttaacgggt 1260ctggctcagg gaagtgggaa gggggtccca gcaagaattc cgtctacatc tcctcgttgt 1320atttcaccat gaccagcctc accagcgtgg gctttgggaa catcgccccg tccacagaca 1380ttgagaagat ctttgccgtg gccatcatga tgattggctc cctcctctat gccaccatct 1440ttgggaatgt gacgaccatt ttccaacaga tgtacgccaa caccaacagg taccatgaga 1500tgctcaacag tgtccgggac ttcttgaagc tctaccaggt gcccaagggg ctgagcgagc 1560gagtcatgga ttacatcgtg tccacctggt ccatgtccag aggcattgac acagagaagg 1620tcctgcagat ctgccccaag gacatgagag cggacatctg cgtgcaccta aaccgcaagg 1680tcttcaagga gcacccagcc tttcggctgg ccagcgacgg ctgcctgcgg gcactggcca 1740tggagttcca gacggtgcac tgcgcccctg gggacctcat ctaccacgca ggggagagcg 1800tcgacagcct gtgcttcgtg gtctccggct ccctggaggt gatccaggat gacgaggtgg 1860tggccattct agggaaagga gacgtgtttg gagacgtgtt ctggaaggaa gccacccttg 1920cccagtcctg tgccaatgtg agggccttga cctactgtga cctccatgtg atcaagcggg 1980acgccctgca gaaagtgctg gaattctaca cagccttctc ccactccttc tcccggaacc 2040tcattctcac ctacaacttg aggaagcgga tcgtgttccg gaagatcagt gacgtgaaac 2100gggaggagga ggagcgcatg aagcggaaga atgaggcccc cctgatcctg ccgcccgacc 2160accccgtccg gcggctcttc cagaggttcc gccagcagaa ggaagccagg ctggccgcgg 2220agaggggcgg gcgggacttg gacgacctgg acgtggagaa gggcagcgtc ctcaccgagc 2280acagccacca cggcctggcg aaggccagcg tcgtcaccgt ccgagagagc cctgccacgc 2340ccgtggcctt cccggcggcc gctgccccgg cggggctgga tcacgcccgg ctgcaggcgc 2400ctggggccga gggcctgggc cccaaggccg gcggggccga ctgcgccaag cgcaagggct 2460gggcccgctt caaggatgcc tgcgggcagg ctgaggactg gagcaaggtg tccaaggccg 2520agtccatgga aacgctcccc gagaggacga aggccgccgg cgaggccaca ctcaagaaga 2580cggactcgtg cgacagcggc atcaccaaga gcgacctgcg tctggacaac gtgggcgagg 2640ccagaagccc ccaggaccgg agccccatct tggcggaggt caagcactcc ttctacccca 2700tccccgagca gacgctgcag gccgccgtcc tggaggtgaa gcacgagctc aaggaggaca 2760tcaaggcctt gagcaccaag atgacgagca ttgagaaaca gctctctgag atactcagga 2820tattaacctc cagaagatcc tctcagtcgc ctcaggagct atttgaaata tcgaggcccc 2880agtccccaga gtcagagaga gacatttttg gcgcaagctg agaggtctgt tgtaaaaaaa 2940aagaaaaaaa atccaagatg acaaaaacct accgtcctgc cctagacacc accacacaca 3000cacctacatg accaacaacc ttcaaagtag gcttttccca a 3041 20 3041 DNA Rattussp. 20 tgcggtgaga cacggcgccg gacgccccca gagccccagc agtagggagg atgaccatgg60 ctggcggccg gcggggacta gtggccccgc agaacacatt tctggagaac atcgtgcggc 120ggtccaacga cactaatttt gtgttgggga atgcccagat cgtggactgg cccatcgtgt 180acagcaatga tggattctgc aagctgtctg gctaccaccg agcggaagtg atgcaaaaga 240gtagcgcctg cagttttatg tatggagagc tgaccgacaa ggacacggtt gaaaaggttc 300gccagacctt tgagaactac gagatgaact ccttcgaaat tctgatgtac aagaagaaca 360ggacacctgt gtggtttttt gtgaagatcg ctccaatcag gaacgaacag gataaagtgg 420ttctgttcct ttgcactttc agtgacataa cggcattcaa gcagcccatt gaggacgact 480cctgcaaagg ttgggggaag tttgctcgac tgacgagagc tctgacaagc agcaggggag 540tcctgcagca gctggccccc agtgtgcaga agggtgagaa tgttcacaag cactcgcgcc 600tggcagaggt cctgcagctg ggttcagaca tcctccccca gtacaagcaa gaggcgccaa 660agacaccccc tcacatcatc ctacactact gtgtctttaa gaccacatgg gattggatca 720tcttgatcct gaccttctac acagccatcc tggtccctta caacgtctcc tttaaaacca 780ggcagaataa cgtggcctgg ctggtggtgg acagcatcgt ggatgtcatc tttttggtgg 840acattgtctt gaattttcac accacctttg tcgggccagc gggggaagtg atctctgacc 900ccaaacttat ccgcatgaac tacctgaaga cgtggtttgt gatcgacctt ctctcctgtt 960tgccatatga cgtcatcaac gcttttgaga acgtggatga gggcatcagc agcctgttca 1020gttctctgaa agtcgtgcgg ctgctccgtc tcggacgagt ggcccgcaag ctggaccatt 1080atatcgagta cggagcggcg gtactggtcc tgctggtgtg cgtgttcggg ctggctgccc 1140actggatggc ctgcatctgg tacagcattg gggattatga gatctttgat gaagacacca 1200agaccatccg taacaacagc tggctctacc aactggcatt ggacattggc actccatacc 1260agtttaatgg gtctggttcg gggaagtggg aaggcgggcc aagcaagaac tccgtataca 1320tttcctcgct gtacttcacc atgacaagtc tcaccagtgt gggctttggt aacatcgccc 1380catccacaga catcgagaag atcttcgccg tagccatcat gatgattggc tcccttctgt 1440atgccaccat ctttgggaat gtgacgacca ttttccagca gatgtatgcc aacaccaaca 1500ggtatcatga gatgctcaac agcgtccggg atttcctgaa gctctaccag gtgcccaagg 1560ggctgagcga gcgggtcatg gactacattg tgtctacctg gtccatgtcc cgcggcatcg 1620acacggagaa ggtcctgcaa atctgcccca aggacatgcg agctgacatt tgcgtacacc 1680tgaaccgaaa agtgttcaaa gaacaccccg ccttccggct ggccagcgat ggttgcctga 1740gggccttggc catggagttc cagacagtac actgcgcccc aggggacctc atctatcacg 1800ccggggagag tgtggacagc ctctgcttcg tggtctcggg ctccctggag gtgatccagg 1860atgatgaggt ggtggccatc ctagggaaag gagatgtgtt tggggatgtt ttctggaagg 1920aggctaccct tgcacagtcc tgcgctaatg tccgggcctt gacctactgt gacctgcacg 1980tgatcaagag ggatgccctg cagaaagtgc tagaattcta cacagccttc tcccactcct 2040tctcccggaa cctgattctc acctacaatc tgaggaagag gattgtgttc cggaagatca 2100gcgacgtgaa acgagaagaa gaggagagga tgaaacggaa gaacgaggcc ccccttatcc 2160tgcctcctga ccaccctgtc aggaggctct tccaaaggtt ccgccagcag aaagaagcca 2220ggctggcagc cgagagaggt ggccgggacc tggatgacct ggatgtagag aagggcaatg 2280ccctcacgga ccatacctca gccaaccaca gcctggtgaa ggccagtgtg gtcacggtgc 2340gtgagagtcc cgccacgcct gtgtccttcc aggcagcctc cacctccaca gtgtcagacc 2400acgccaagct gcatgcaccg ggatctgagt gcctaggtcc caaggcaggc ggtggcgacc 2460ctgccaagcg caaaggctgg gcccggttca aagatgcctg tgggaagggt gaggattgga 2520acaaggtgtc caaggcagag tccatggaga cgcttcccga gaggacaaag gcatcgggcg 2580aggccacgct gaagaagaca gactcctgtg acagtggaat caccaagagt gacctgcgct 2640tggacaatgt gggtgaggcc aggagtcccc aggaccggag ccccatcttg gccgaggtca 2700agcattcttt ctaccccatc cccgagcaga cactgcaggc cacagtgctg gaggtgaagc 2760atgagctgaa ggaagacatc aaggccttga atgccaaaat gacctccatt gagaagcagc 2820tgtctgagat cctcaggata ctcatgtcca gagggtcctc ccagtctccg caggacacgt 2880gtgaggtctc caggccccag tccccagagt cagacagaga catttttggg gcaagctgag 2940aggatcattt caaaacaaac aaacaaaaaa atcaaagaca aaagcctgcc ccctgcccct 3000gacacttcct accgcaccaa acacatgacc aacaactttc a 3041 21 960 PRT Bovine sp.21 Met Thr Met Ala Gly Gly Arg Lys Gly Leu Val Ala Pro Gln Asn Thr 1 510 15 Phe Leu Glu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 2025 30 Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly 3540 45 Phe Cys Lys Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 5055 60 Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Ile 6570 75 80 Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu85 90 95 Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Phe Val Lys100 105 110 Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe LeuCys 115 120 125 Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu AspAsp Ser 130 135 140 Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg AlaLeu Thr Ser 145 150 155 160 Ser Arg Gly Val Leu Gln Gln Leu Ala Pro SerVal Gln Lys Gly Glu 165 170 175 Asn Val His Lys His Ser Arg Leu Ala GluVal Leu Gln Leu Gly Ser 180 185 190 Asp Ile Leu Pro Gln Tyr Lys Gln GluAla Pro Lys Thr Pro Pro His 195 200 205 Ile Ile Leu His Tyr Cys Val PheLys Thr Thr Trp Asp Trp Ile Ile 210 215 220 Leu Ile Leu Thr Phe Tyr ThrAla Ile Leu Val Pro Tyr Asn Val Ser 225 230 235 240 Phe Lys Thr Arg GlnAsn Asn Val Ala Trp Leu Val Val Asp Ser Ile 245 250 255 Val Asp Val IlePhe Leu Val Asp Ile Val Leu Asn Phe His Thr Thr 260 265 270 Phe Val GlyPro Ala Gly Glu Val Ile Ser Asp Pro Lys Leu Ile Arg 275 280 285 Met AsnTyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290 295 300 ProTyr Asp Val Ile Asn Ala Phe Glu Asn Val Asp Glu Gly Ile Ser 305 310 315320 Ser Leu Phe Ser Ser Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg 325330 335 Val Ala Arg Lys Leu Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu340 345 350 Val Leu Leu Val Cys Val Phe Gly Leu Ala Ala His Trp Met AlaCys 355 360 365 Ile Trp Tyr Ser Ile Gly Asp Tyr Glu Ile Phe Asp Glu AspThr Lys 370 375 380 Thr Ile Arg Asn Asn Ser Trp Leu Tyr Gln Leu Ala MetAsp Ile Gly 385 390 395 400 Thr Pro Tyr Gln Phe Asn Gly Ser Gly Ser GlyLys Trp Glu Gly Gly 405 410 415 Pro Ser Lys Asn Ser Val Tyr Ile Ser SerLeu Tyr Phe Thr Met Thr 420 425 430 Ser Leu Thr Ser Val Gly Phe Gly AsnIle Ala Pro Ser Thr Asp Ile 435 440 445 Glu Lys Ile Phe Ala Val Ala IleMet Met Ile Gly Ser Leu Leu Tyr 450 455 460 Ala Thr Ile Phe Gly Asn ValThr Thr Ile Phe Gln Gln Met Tyr Ala 465 470 475 480 Asn Thr Asn Arg TyrHis Glu Met Leu Asn Ser Val Arg Asp Phe Leu 485 490 495 Lys Leu Tyr GlnVal Pro Lys Gly Leu Ser Glu Arg Val Met Asp Tyr 500 505 510 Ile Val SerThr Trp Ser Met Ser Arg Gly Ile Asp Thr Glu Lys Val 515 520 525 Leu GlnIle Cys Pro Lys Asp Met Arg Ala Asp Ile Cys Val His Leu 530 535 540 AsnArg Lys Val Phe Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp 545 550 555560 Gly Cys Leu Arg Ala Leu Ala Met Glu Phe Gln Thr Val His Cys Ala 565570 575 Pro Gly Asp Leu Ile Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys580 585 590 Phe Val Val Ser Gly Ser Leu Glu Val Ile Gln Asp Asp Glu ValVal 595 600 605 Ala Ile Leu Gly Lys Gly Asp Val Phe Gly Asp Val Phe TrpLys Glu 610 615 620 Ala Thr Leu Ala Gln Ser Cys Ala Asn Val Arg Ala LeuThr Tyr Cys 625 630 635 640 Asp Leu His Val Ile Lys Arg Asp Ala Leu GlnLys Val Leu Glu Phe 645 650 655 Tyr Thr Ala Phe Ser His Ser Phe Ser ArgAsn Leu Ile Leu Thr Tyr 660 665 670 Asn Leu Arg Lys Arg Ile Val Phe ArgLys Ile Ser Asp Val Lys Arg 675 680 685 Glu Glu Glu Glu Arg Met Lys ArgLys Asn Glu Ala Pro Leu Ile Leu 690 695 700 Pro Pro Asp His Pro Val ArgArg Leu Phe Gln Arg Phe Arg Gln Gln 705 710 715 720 Lys Glu Ala Arg LeuAla Ala Glu Arg Gly Gly Arg Asp Leu Asp Asp 725 730 735 Leu Asp Val GluLys Gly Ser Val Leu Thr Glu His Ser His His Gly 740 745 750 Leu Ala LysAla Ser Val Val Thr Val Arg Glu Ser Pro Ala Thr Pro 755 760 765 Val AlaPhe Pro Ala Ala Ala Ala Pro Ala Gly Leu Asp His Ala Arg 770 775 780 LeuGln Ala Pro Gly Ala Glu Gly Leu Gly Pro Lys Ala Gly Gly Ala 785 790 795800 Asp Cys Ala Lys Arg Lys Gly Trp Ala Arg Phe Lys Asp Ala Cys Gly 805810 815 Gln Ala Glu Asp Trp Ser Lys Val Ser Lys Ala Glu Ser Met Glu Thr820 825 830 Leu Pro Glu Arg Thr Lys Ala Ala Gly Glu Ala Thr Leu Lys LysThr 835 840 845 Asp Ser Cys Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg LeuAsp Asn 850 855 860 Val Gly Glu Ala Arg Ser Pro Gln Asp Arg Ser Pro IleLeu Ala Glu 865 870 875 880 Val Lys His Ser Phe Tyr Pro Ile Pro Glu GlnThr Leu Gln Ala Ala 885 890 895 Val Leu Glu Val Lys His Glu Leu Lys GluAsp Ile Lys Ala Leu Ser 900 905 910 Thr Lys Met Thr Ser Ile Glu Lys GlnLeu Ser Glu Ile Leu Arg Ile 915 920 925 Leu Thr Ser Arg Arg Ser Ser GlnSer Pro Gln Glu Leu Phe Glu Ile 930 935 940 Ser Arg Pro Gln Ser Pro GluSer Glu Arg Asp Ile Phe Gly Ala Ser 945 950 955 960 22 987 PRT Bovinesp. 22 Met Thr Met Ala Gly Gly Arg Lys Gly Leu Val Ala Pro Gln Asn Thr 15 10 15 Phe Leu Glu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn Phe Val Leu20 25 30 Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly35 40 45 Phe Cys Lys Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser50 55 60 Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Ile65 70 75 80 Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser PheGlu 85 90 95 Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Phe ValLys 100 105 110 Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu PheLeu Cys 115 120 125 Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile GluAsp Asp Ser 130 135 140 Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr ArgAla Leu Thr Ser 145 150 155 160 Ser Arg Gly Val Leu Gln Gln Leu Ala ProSer Val Gln Lys Gly Glu 165 170 175 Asn Val His Lys His Ser Arg Leu AlaGlu Val Leu Gln Leu Gly Ser 180 185 190 Asp Ile Leu Pro Gln Tyr Lys GlnGlu Ala Pro Lys Thr Pro Pro His 195 200 205 Ile Ile Leu His Tyr Cys ValPhe Lys Thr Thr Trp Asp Trp Ile Ile 210 215 220 Leu Ile Leu Thr Phe TyrThr Ala Ile Leu Val Pro Tyr Asn Val Ser 225 230 235 240 Phe Lys Thr ArgGln Asn Asn Val Ala Trp Leu Val Val Asp Ser Ile 245 250 255 Val Asp ValIle Phe Leu Val Asp Ile Val Leu Asn Phe His Thr Thr 260 265 270 Phe ValGly Pro Ala Gly Glu Val Ile Ser Asp Pro Lys Leu Ile Arg 275 280 285 MetAsn Tyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290 295 300Pro Tyr Asp Val Ile Asn Ala Phe Glu Asn Val Asp Glu Val Ser Ala 305 310315 320 Phe Met Gly Asp Pro Gly Lys Ile Gly Phe Ala Asp Gln Ile Pro Pro325 330 335 Pro Leu Glu Gly Arg Glu Ser Gln Gly Ile Ser Ser Leu Phe SerSer 340 345 350 Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg Val Ala ArgLys Leu 355 360 365 Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu Val LeuLeu Val Cys 370 375 380 Val Phe Gly Leu Ala Ala His Trp Met Ala Cys IleTrp Tyr Ser Ile 385 390 395 400 Gly Asp Tyr Glu Ile Phe Asp Glu Asp ThrLys Thr Ile Arg Asn Asn 405 410 415 Ser Trp Leu Tyr Gln Leu Ala Met AspIle Gly Thr Pro Tyr Gln Phe 420 425 430 Asn Gly Ser Gly Ser Gly Lys TrpGlu Gly Gly Pro Ser Lys Asn Ser 435 440 445 Val Tyr Ile Ser Ser Leu TyrPhe Thr Met Thr Ser Leu Thr Ser Val 450 455 460 Gly Phe Gly Asn Ile AlaPro Ser Thr Asp Ile Glu Lys Ile Phe Ala 465 470 475 480 Val Ala Ile MetMet Ile Gly Ser Leu Leu Tyr Ala Thr Ile Phe Gly 485 490 495 Asn Val ThrThr Ile Phe Gln Gln Met Tyr Ala Asn Thr Asn Arg Tyr 500 505 510 His GluMet Leu Asn Ser Val Arg Asp Phe Leu Lys Leu Tyr Gln Val 515 520 525 ProLys Gly Leu Ser Glu Arg Val Met Asp Tyr Ile Val Ser Thr Trp 530 535 540Ser Met Ser Arg Gly Ile Asp Thr Glu Lys Val Leu Gln Ile Cys Pro 545 550555 560 Lys Asp Met Arg Ala Asp Ile Cys Val His Leu Asn Arg Lys Val Phe565 570 575 Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp Gly Cys Leu ArgAla 580 585 590 Leu Ala Met Glu Phe Gln Thr Val His Cys Ala Pro Gly AspLeu Ile 595 600 605 Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys Phe ValVal Ser Gly 610 615 620 Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val AlaIle Leu Gly Lys 625 630 635 640 Gly Asp Val Phe Gly Asp Val Phe Trp LysGlu Ala Thr Leu Ala Gln 645 650 655 Ser Cys Ala Asn Val Arg Ala Leu ThrTyr Cys Asp Leu His Val Ile 660 665 670 Lys Arg Asp Ala Leu Gln Lys ValLeu Glu Phe Tyr Thr Ala Phe Ser 675 680 685 His Ser Phe Ser Arg Asn LeuIle Leu Thr Tyr Asn Leu Arg Lys Arg 690 695 700 Ile Val Phe Arg Lys IleSer Asp Val Lys Arg Glu Glu Glu Glu Arg 705 710 715 720 Met Lys Arg LysAsn Glu Ala Pro Leu Ile Leu Pro Pro Asp His Pro 725 730 735 Val Arg ArgLeu Phe Gln Arg Phe Arg Gln Gln Lys Glu Ala Arg Leu 740 745 750 Ala AlaGlu Arg Gly Gly Arg Asp Leu Asp Asp Leu Asp Val Glu Lys 755 760 765 GlySer Val Leu Thr Glu His Ser His His Gly Leu Ala Lys Ala Ser 770 775 780Val Val Thr Val Arg Glu Ser Pro Ala Thr Pro Val Ala Phe Pro Ala 785 790795 800 Ala Ala Ala Pro Ala Gly Leu Asp His Ala Arg Leu Gln Ala Pro Gly805 810 815 Ala Glu Gly Leu Gly Pro Lys Ala Gly Gly Ala Asp Cys Ala LysArg 820 825 830 Lys Gly Trp Ala Arg Phe Lys Asp Ala Cys Gly Gln Ala GluAsp Trp 835 840 845 Ser Lys Val Ser Lys Ala Glu Ser Met Glu Thr Leu ProGlu Arg Thr 850 855 860 Lys Ala Ala Gly Glu Ala Thr Leu Lys Lys Thr AspSer Cys Asp Ser 865 870 875 880 Gly Ile Thr Lys Ser Asp Leu Arg Leu AspAsn Val Gly Glu Ala Arg 885 890 895 Ser Pro Gln Asp Arg Ser Pro Ile LeuAla Glu Val Lys His Ser Phe 900 905 910 Tyr Pro Ile Pro Glu Gln Thr LeuGln Ala Ala Val Leu Glu Val Lys 915 920 925 His Glu Leu Lys Glu Asp IleLys Ala Leu Ser Thr Lys Met Thr Ser 930 935 940 Ile Glu Lys Gln Leu SerGlu Ile Leu Arg Ile Leu Thr Ser Arg Arg 945 950 955 960 Ser Ser Gln SerPro Gln Glu Leu Phe Glu Ile Ser Arg Pro Gln Ser 965 970 975 Pro Glu SerGlu Arg Asp Ile Phe Gly Ala Ser 980 985 23 989 PRT Mus sp. 23 Met ThrMet Ala Gly Gly Arg Lys Gly Leu Val Ala Pro Gln Asn Thr 1 5 10 15 PheLeu Glu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 20 25 30 GlyAsn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly 35 40 45 PheCys Lys Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 50 55 60 SerAla Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Val 65 70 75 80Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu 85 90 95Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Phe Val Lys 100 105110 Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe Leu Cys 115120 125 Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu Asp Asp Ser130 135 140 Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg Ala Leu ThrSer 145 150 155 160 Ser Arg Gly Val Leu Gln Gln Leu Ala Pro Ser Val GlnLys Gly Glu 165 170 175 Asn Val His Lys His Ser Arg Leu Ala Glu Val LeuGln Leu Gly Ser 180 185 190 Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala ProLys Thr Pro Pro His 195 200 205 Ile Ile Leu His Tyr Cys Val Phe Lys ThrThr Trp Asp Trp Ile Ile 210 215 220 Leu Ile Leu Thr Phe Tyr Thr Ala IleLeu Val Pro Tyr Asn Val Ser 225 230 235 240 Phe Lys Thr Arg Gln Asn AsnVal Ala Trp Leu Val Val Asp Ser Ile 245 250 255 Val Asp Val Ile Phe LeuVal Asp Ile Val Leu Asn Phe His Thr Thr 260 265 270 Phe Val Gly Pro AlaGly Glu Val Ile Ser Asp Pro Lys Leu Ile Arg 275 280 285 Met Asn Tyr LeuLys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290 295 300 Pro Tyr AspVal Ile Asn Ala Phe Glu Asn Val Asp Glu Val Ser Ala 305 310 315 320 PheMet Gly Asp Pro Gly Lys Ile Gly Phe Ala Asp Gln Ile Pro Pro 325 330 335Pro Leu Glu Gly Arg Glu Ser Gln Gly Ile Ser Ser Leu Phe Ser Ser 340 345350 Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg Val Ala Arg Lys Leu 355360 365 Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu Val Leu Leu Val Cys370 375 380 Val Phe Gly Leu Ala Ala His Trp Met Ala Cys Ile Trp Tyr SerIle 385 390 395 400 Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr Lys Thr IleArg Asn Asn 405 410 415 Ser Trp Leu Tyr Gln Leu Ala Leu Asp Ile Gly ThrPro Tyr Gln Phe 420 425 430 Asn Gly Ser Gly Ser Gly Lys Trp Glu Gly GlyPro Ser Lys Asn Ser 435 440 445 Val Tyr Ile Ser Ser Leu Tyr Phe Thr MetThr Ser Leu Thr Ser Val 450 455 460 Gly Phe Gly Asn Ile Ala Pro Ser ThrAsp Ile Glu Lys Ile Phe Ala 465 470 475 480 Val Ala Ile Met Met Ile GlySer Leu Leu Tyr Ala Thr Ile Phe Gly 485 490 495 Asn Val Thr Thr Ile PheGln Gln Met Tyr Ala Asn Thr Asn Arg Tyr 500 505 510 His Glu Met Leu AsnSer Val Arg Asp Phe Leu Lys Leu Tyr Gln Val 515 520 525 Pro Lys Gly LeuSer Glu Arg Val Met Asp Tyr Ile Val Ser Thr Trp 530 535 540 Ser Met SerArg Gly Ile Asp Thr Glu Lys Val Leu Gln Ile Cys Pro 545 550 555 560 LysAsp Met Arg Ala Asp Ile Cys Val His Leu Asn Arg Lys Val Phe 565 570 575Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp Gly Cys Leu Arg Ala 580 585590 Leu Ala Met Glu Phe Gln Thr Val His Cys Ala Pro Gly Asp Leu Ile 595600 605 Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys Phe Val Val Ser Gly610 615 620 Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val Ala Ile Leu GlyLys 625 630 635 640 Gly Asp Val Phe Gly Asp Val Phe Trp Lys Glu Ala ThrLeu Ala Gln 645 650 655 Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr Cys AspLeu His Val Ile 660 665 670 Lys Arg Asp Ala Leu Gln Lys Val Leu Glu PheTyr Thr Ala Phe Ser 675 680 685 His Ser Phe Ser Arg Asn Leu Ile Leu ThrTyr Asn Leu Arg Lys Arg 690 695 700 Ile Val Phe Arg Lys Ile Ser Asp ValLys Arg Glu Glu Glu Glu Arg 705 710 715 720 Met Lys Arg Lys Asn Glu AlaPro Leu Ile Leu Pro Pro Asp His Pro 725 730 735 Val Arg Arg Leu Phe GlnArg Phe Arg Gln Gln Lys Glu Ala Arg Leu 740 745 750 Ala Ala Glu Arg GlyGly Arg Asp Leu Asp Asp Leu Asp Val Glu Lys 755 760 765 Gly Asn Ala LeuThr Asp His Thr Ser Ala Asn His Gly Leu Ala Lys 770 775 780 Ala Ser ValVal Thr Val Arg Glu Ser Pro Ala Thr Pro Val Ala Phe 785 790 795 800 GlnAla Ala Thr Thr Ser Thr Met Ser Asp His Ala Lys Leu His Ala 805 810 815Pro Gly Ser Glu Cys Leu Gly Pro Lys Ala Val Ser Cys Asp Pro Ala 820 825830 Lys Arg Lys Gly Trp Ala Arg Phe Lys Asp Ala Cys Gly Gln Ala Glu 835840 845 Asp Trp Ser Lys Val Ser Lys Ala Glu Ser Met Glu Thr Leu Pro Glu850 855 860 Arg Thr Lys Ala Pro Gly Glu Ala Thr Leu Lys Lys Thr Asp SerCys 865 870 875 880 Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu Asp AsnVal Gly Glu 885 890 895 Thr Arg Ser Pro Gln Asp Arg Ser Pro Ile Leu AlaGlu Val Lys His 900 905 910 Ser Phe Tyr Pro Ile Pro Glu Gln Thr Leu GlnAla Ala Val Leu Glu 915 920 925 Val Lys Tyr Glu Leu Lys Glu Asp Ile LysAla Leu Asn Ala Lys Met 930 935 940 Thr Ser Ile Glu Lys Gln Leu Ser GluIle Leu Arg Ile Leu Met Ser 945 950 955 960 Arg Gly Ser Ala Gln Ser ProGln Glu Thr Gly Glu Ile Ser Arg Pro 965 970 975 Gln Ser Pro Glu Ser AspArg Asp Ile Phe Gly Ala Ser 980 985 24 962 PRT Rattus sp. 24 Met Thr MetAla Gly Gly Arg Lys Gly Leu Val Ala Pro Gln Asn Thr 1 5 10 15 Phe LeuGlu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn Phe Val Leu 20 25 30 Gly AsnAla Gln Ile Val Asp Trp Pro Ile Val Tyr Ser Asn Asp Gly 35 40 45 Phe CysLys Leu Ser Gly Tyr His Arg Ala Glu Val Met Gln Lys Ser 50 55 60 Ser AlaCys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Asp Thr Val 65 70 75 80 GluLys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met Asn Ser Phe Glu 85 90 95 IleLeu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Phe Val Lys 100 105 110Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val Val Leu Phe Leu Cys 115 120125 Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln Pro Ile Glu Asp Asp Ser 130135 140 Cys Lys Gly Trp Gly Lys Phe Ala Arg Leu Thr Arg Ala Leu Thr Ser145 150 155 160 Ser Arg Gly Val Leu Gln Gln Leu Ala Pro Ser Val Gln LysGly Glu 165 170 175 Asn Val His Lys His Ser Arg Leu Ala Glu Val Leu GlnLeu Gly Ser 180 185 190 Asp Ile Leu Pro Gln Tyr Lys Gln Glu Ala Pro LysThr Pro Pro His 195 200 205 Ile Ile Leu His Tyr Cys Val Phe Lys Thr ThrTrp Asp Trp Ile Ile 210 215 220 Leu Ile Leu Thr Phe Tyr Thr Ala Ile LeuVal Pro Tyr Asn Val Ser 225 230 235 240 Phe Lys Thr Arg Gln Asn Asn ValAla Trp Leu Val Val Asp Ser Ile 245 250 255 Val Asp Val Ile Phe Leu ValAsp Ile Val Leu Asn Phe His Thr Thr 260 265 270 Phe Val Gly Pro Ala GlyGlu Val Ile Ser Asp Pro Lys Leu Ile Arg 275 280 285 Met Asn Tyr Leu LysThr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290 295 300 Pro Tyr Asp ValIle Asn Ala Phe Glu Asn Val Asp Glu Gly Ile Ser 305 310 315 320 Ser LeuPhe Ser Ser Leu Lys Val Val Arg Leu Leu Arg Leu Gly Arg 325 330 335 ValAla Arg Lys Leu Asp His Tyr Ile Glu Tyr Gly Ala Ala Val Leu 340 345 350Val Leu Leu Val Cys Val Phe Gly Leu Ala Ala His Trp Met Ala Cys 355 360365 Ile Trp Tyr Ser Ile Gly Asp Tyr Glu Ile Phe Asp Glu Asp Thr Lys 370375 380 Thr Ile Arg Asn Asn Ser Trp Leu Tyr Gln Leu Ala Leu Asp Ile Gly385 390 395 400 Thr Pro Tyr Gln Phe Asn Gly Ser Gly Ser Gly Lys Trp GluGly Gly 405 410 415 Pro Ser Lys Asn Ser Val Tyr Ile Ser Ser Leu Tyr PheThr Met Thr 420 425 430 Ser Leu Thr Ser Val Gly Phe Gly Asn Ile Ala ProSer Thr Asp Ile 435 440 445 Glu Lys Ile Phe Ala Val Ala Ile Met Met IleGly Ser Leu Leu Tyr 450 455 460 Ala Thr Ile Phe Gly Asn Val Thr Thr IlePhe Gln Gln Met Tyr Ala 465 470 475 480 Asn Thr Asn Arg Tyr His Glu MetLeu Asn Ser Val Arg Asp Phe Leu 485 490 495 Lys Leu Tyr Gln Val Pro LysGly Leu Ser Glu Arg Val Met Asp Tyr 500 505 510 Ile Val Ser Thr Trp SerMet Ser Arg Gly Ile Asp Thr Glu Lys Val 515 520 525 Leu Gln Ile Cys ProLys Asp Met Arg Ala Asp Ile Cys Val His Leu 530 535 540 Asn Arg Lys ValPhe Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp 545 550 555 560 Gly CysLeu Arg Ala Leu Ala Met Glu Phe Gln Thr Val His Cys Ala 565 570 575 ProGly Asp Leu Ile Tyr His Ala Gly Glu Ser Val Asp Ser Leu Cys 580 585 590Phe Val Val Ser Gly Ser Leu Glu Val Ile Gln Asp Asp Glu Val Val 595 600605 Ala Ile Leu Gly Lys Gly Asp Val Phe Gly Asp Val Phe Trp Lys Glu 610615 620 Ala Thr Leu Ala Gln Ser Cys Ala Asn Val Arg Ala Leu Thr Tyr Cys625 630 635 640 Asp Leu His Val Ile Lys Arg Asp Ala Leu Gln Lys Val LeuGlu Phe 645 650 655 Tyr Thr Ala Phe Ser His Ser Phe Ser Arg Asn Leu IleLeu Thr Tyr 660 665 670 Asn Leu Arg Lys Arg Ile Val Phe Arg Lys Ile SerAsp Val Lys Arg 675 680 685 Glu Glu Glu Glu Arg Met Lys Arg Lys Asn GluAla Pro Leu Ile Leu 690 695 700 Pro Pro Asp His Pro Val Arg Arg Leu PheGln Arg Phe Arg Gln Gln 705 710 715 720 Lys Glu Ala Arg Leu Ala Ala GluArg Gly Gly Arg Asp Leu Asp Asp 725 730 735 Leu Asp Val Glu Lys Gly AsnAla Leu Thr Asp His Thr Ser Ala Asn 740 745 750 His Gly Leu Ala Lys AlaSer Val Val Thr Val Arg Glu Ser Pro Ala 755 760 765 Thr Pro Val Ala PheGln Ala Ala Ser Thr Ser Thr Val Ser Asp His 770 775 780 Ala Lys Leu HisAla Pro Gly Ser Glu Cys Leu Gly Pro Lys Ala Gly 785 790 795 800 Gly GlyAsp Pro Ala Lys Arg Lys Gly Trp Ala Arg Phe Lys Asp Ala 805 810 815 CysGly Gln Ala Glu Asp Trp Ser Lys Val Ser Lys Ala Glu Ser Met 820 825 830Glu Thr Leu Pro Glu Arg Thr Lys Ala Ala Gly Glu Ala Thr Leu Lys 835 840845 Lys Thr Asp Ser Cys Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu 850855 860 Asp Asn Val Gly Glu Ala Arg Ser Pro Gln Asp Arg Ser Pro Ile Leu865 870 875 880 Ala Glu Val Lys His Ser Phe Tyr Pro Ile Pro Glu Gln ThrLeu Gln 885 890 895 Ala Thr Val Leu Glu Val Lys Tyr Glu Leu Lys Glu AspIle Lys Ala 900 905 910 Leu Asn Ala Lys Met Thr Ser Ile Glu Lys Gln LeuSer Glu Ile Leu 915 920 925 Arg Ile Leu Met Ser Arg Gly Ser Ser Gln SerPro Gln Asp Thr Cys 930 935 940 Glu Val Ser Arg Pro Gln Ser Pro Glu SerAsp Arg Asp Ile Phe Gly 945 950 955 960 Ala Ser

1. A nucleic acid molecule comprising a nucleic acid molecule encoding a(poly)peptide having a function of the human K⁺ ion eag channel which is(a) a nucleic acid molecule comprising a nucleic acid molecule encodingthe polypeptide having the amino acid sequence of SEQ ID: No 3 or 4; (b)a nucleic acid molecule comprising the nucleic acid molecule having theDNA sequence of SEQ ID: No 13 or 14; (c) a nucleic acid moleculehybridizing to the complementary strand of a nucleic acid molecule of(a) or (b); or (d) a nucleic acid molecule being degenerate to thesequence of the nucleic acid molecule of (c).
 2. A nucleic acid moleculespecifically hybridizing to the nucleic acid molecule of claim 1 whichcomprises the sequence 5′-GGGAGGATGACCATGGCT.
 3. The nucleic acidmolecule of claim 1 or 2 which is DNA.
 4. The nucleic acid molecule ofclaim 1 or 2 which is RNA.
 5. The nucleic acid molecule of any one ofclaims 1 to 4 encoding a fusion protein.
 6. A vector comprising thenucleic acid molecule of any one of claims 1 to
 5. 7. The vector ofclaim 6 which is an expression vector and/or a gene targeting or genetransfer vector.
 8. A host transformed with a vector of claim 6 or
 7. 9.The host of claim 8 which is a mammalian cell, a fungal cell, a plantcell, an insect cell or a bacterial cell.
 10. A method of producing the(poly)peptide encoded by the nucleic acid molecule of any one of claims1 or 3 to 5 comprising culturing the host of claim 8 or 9 and isolatingthe produced (poly)peptide.
 11. A (poly)peptide encoded by the nucleicacid of any one of claims 1 or 3 to 5 or produced by the method of claim10.
 12. An antibody specifically directed to the (poly)peptide of claim11.
 13. The antibody of claim 12 which is a monoclonal antibody.
 14. Apharmaceutical composition comprising the nucleic acid molecule of claim2, the vector of claim 6, the polypeptide of claim 11 and/or theantibody of claim 12 or 13 and a pharmaceutically acceptable carrierand/or diluent and/or excipient.
 15. A diagnostic composition comprisingthe nucleic acid molecule of any one of claims 1 to 5, the vector ofclaim 6, the polypeptide of claim 11 and/or the antibody of claim 12 or13.
 16. A method for preventing or treating a disease which is caused bythe undesired expression or overexpression of the nucleic acid moleculeof any one of claims 1 or 3 to 5, comprising introducing an inhibitor ofthe expression of the nucleic acid molecule of any one of claims 1 or 3to 5 or an inhibitor of function of the (poly)peptide of claim 11 into amammal affected by said disease or being suspected of being susceptibleto said disease.
 17. A method for preventing or treating a disease whichis caused by the malfunction of the (poly)peptide of claim 11 comprisingintroducing an inhibitor of the expression of the nucleic acid moleculeof any one of claims 1 or 3 to 5 or an inhibitor or modifying agent ofthe malfunction of the (poly)peptide of claim 11 or the nucleic acidmolecule of any one of claims 1 or 3 to 5 encoding heag or thepolypeptide of claim 11 having heag activity into a mammal affected bysaid disease or being suspected of being susceptible to said disease.18. The method of claim 16 wherein said inhibitor of the expression oroverexpression of said nucleic acid molecule is a nucleic acid moleculeof claim
 2. 19. The method of claim 16 wherein said inhibitor ofpolypeptide function is the antibody of claim 12 or 13 or a drug,preferably astemizole or terfenadine.
 20. The method of any one ofclaims 16 to 19 further comprising, prior to the introduction step, (a)obtaining cells from the mammal infected by said disease and, after saidintroduction step, wherein said introduction is effected into saidcells, (b) reintroducing said cells into said mammal or into a mammal ofthe same species.
 21. The method of any one of claims 16 to 20 whereinsaid cell is a germ cell, an embryonic cell or an egg cell or a cellderived therefrom.
 22. A method of designing a drug for the treatment ofa disease which is caused by the undesired expression or overexpressionof the nucleic acid molecule of any one of claims 1 and 3 to 5comprising (a) identification of a specific and potent drugs; (b)identification of the binding site of said drug by site-directedmutagenesis and chimeric protein studies; (c) molecular modeling of boththe binding site in the (poly)peptide and the structure of said drug;and (c) modifications of the drug to improve its binding specificity forthe (poly)peptide.
 23. A method of identifying an inhibitor of theexpression of the nucleic acid molecule of any one of claims 1 or 3 to 5or an inhibitor of a function of the (poly)peptide of claim 11comprising: (a) testing a compound for the inhibition or reduction oftranslation wherein said compound is selected from antisenseoligonucleotides and/or ribozymes; or (b) testing a compound for theinhibition of transcription wherein said compound binds to the promoterregion of the gene encoding the (poly)peptide of claim 11 and preferablywith transcription factor responsive elements thereof; or (c) testingpeptides or antibodies suspected to block the proliferative activity ofthe (poly)peptide of claim 11 for said blocking activity.
 24. The methodof claim 22 or 23 wherein said drug or inhibitor is further improved bypeptidomimetics or by applying phage-display or combinatorial librarytechniques.
 25. A method of inhibiting cell proliferation comprisingapplying an inhibitor to expression of the nucleic acid of any one ofclaims 1 or 3 to 5 or the (poly)peptide of claim
 11. 26. A method ofprognosing cancer and/or neurodegenerative diseases and/or psoriasiscomprising assessing the expression of the nucleic acid molecule of anyone of claims 1 and 3 to 5 or assessing the quantitative presence of thepolypeptide of claim 11 in cells of a mammal.
 27. The method of claim26, wherein said cancer is mamma carcinoma or neuroblastoma or cervixcarcinoma.
 28. The method of claim 27, wherein said mamma carcinoma isbreast adenocarcinoma, breast carcinoma ductal type.
 29. The method ofclaim 26, wherein said neurodegenerative disease is Alzheimer's disease,Parkinson's disease, lateral amytrophic sclerosis or multiple sclerosis.30. The method of any one of claims 16 to 21 and 26 to 29 wherein saidmammal is a human, rat or mouse.
 31. Use of the nucleic acid molecule ofany one of claims 1 to 5 in gene therapy.
 32. Kit comprising the nucleicacid molecule of claim 2, the vector of claim 6, the polypeptide ofclaim 11 and/or the antibody of claim 12 or 13.